Method for the vaccination against hiv

ABSTRACT

The present invention relates to novel compositions of active agents and methods for the treatment of HIV infection and AIDS. In particular, the present invention relates to novel methods to select HIV infected patients with improved responses to HIV-specific vaccine peptides.

FIELD OF THE INVENTION

The present invention relates to methods for treating reducing and/ordelaying pathological effects of human immunodeficiency virus I (HIV) ina human infected with HIV or for reducing the risk of developingacquired immunodeficiency syndrome (AIDS) in a human.

BACKGROUND OF THE INVENTION

HIV-1 infection is today perceived as an incurable chronic viralinfection in which lifelong combination antiretroviral therapy (cART) isneeded to avoid disease (Egger, Hirschel et al. 1997, Palella, Delaneyet al. 1998). Very early during acute HIV infection a latent reservoiris established and despite effective cART, HIV-1 persists in latentlyinfected cells (Dai, Agosto et al. 2009, Carter, Onafuwa-Nuga et al.2010, Wightman, Solomon et al. 2010). Upon treatment interruption, thevirus quickly replicates, and viremia rebounds to pre-treatment levels.In the inactive, resting state latently infected cells areunrecognizable to the immune system and unresponsive to antiretroviraldrugs (Chun, Stuyver et al. 1997, Finzi, Hermankova et al. 1997). Thesize of the reservoir likely varies between individuals and may beinfluenced by a number of different factors such as host immuneconstitution, time from diagnosis to initiation, level of persistentimmune activation, antiretroviral treatment regimens used and individualresponse to treatment. Earlier studies employing viral outgrowth assaysindicated that the number of latent CD4 T cells harboringreplication-competent virus was approximately 1 per 106 cells.

A broad range of bioanalytical assays have been used in the attempt toquantify the reservoir but it is currently unclear which assay(s) shouldbe used to monitor HIV-1 reservoirs in clinical studies of eradicationstrategies (Eriksson, Graf et al. 2013). Upon activation, resting Tcells carrying replication competent integrated proviral DNA are capableof resuming HIV transcription (Chun, Finzi et al. 1995, Chun, Carruth etal. 1997, Eriksson, Graf et al. 2013). One of the proposed ways ofcuring HIV-1 is to activate and kill latently infected cells in thepresence of antiretroviral therapy (Deeks 2012). Epigenetic modulationof the molecular mechanisms that block transcription of integrated HIVDNA can reactivate HIV-1 expression in resting infected memory CD4 Tcells and disrupt latency (Rasmussen, Schmeltz Sogaard et al. 2013,Rasmussen, Tolstrup et al. 2013). Histone deacetylase inhibitors (HDACi)turn on genes by promoting acetylation of lysine residues on histones(Van Lint, Emiliani et al. 1996, Tyagi, Pearson et al. 2010). Thisinduces chromatin relaxation and transcriptional activation. The HDACiromidepsin (Celgene) potently activates HIV-1 expression in latentlyinfected cell lines and primary T cells (Geleziunas 2013).

Vacc-4x is a peptide-based HIV-1 therapeutic vaccine that aims toimprove immune responses to p24Gag since this has been associated withslower disease progression and improved virus control (Kiepiela 2007;Zuniga 2006). The primary objective of Vacc-4x immunization is tostrengthen the immune system's response to HIV p24. The enhanced immuneresponse to HIV-1 following immunization with Vacc-4x could improve thehost immune system as part of an HIV functional cure treatment strategy.

In one of the largest randomized, placebo controlled HIV therapeuticvaccine trials conducted to date (study CT-BI/Vacc-4x/2007/1), Vacc-4xand rhuGM-CSF (Leukine®) as adjuvant showed a significant reduction inviral load (VL) set point in the Vacc-4x group as compared to placeboand a significant reduction in VL set point from historic preART values,despite higher preART values being present in the Vacc-4x group ascompared to placebo. Additionally Vacc-4x was shown to be immunogenic,inducing proliferative responses in both CD4 and CD8 T-cell.

New HIV p24 peptides are described in WO91/13360, wherein the peptidesare used in a method of discriminating between a false and truediagnosed HIV-positive serum sample.

Johnson R. P., et al., The Journal of Immunology, Vol. 147, p.1512-1521, No. 5, Sep. 1, 1991 describe an analysis of the finespecificity of gag-specific CTL-responses in three HIV-1 seropositiveindividuals, the gag-specific CTL-responses were found to be mediated byCD3+CD8+ lymphocytes which are HLA class I restricted.

EP-A-0 356 007 discloses antigenic determinants, in particular itrelates to synthetic polypeptide sequences which are related to proteinspresent in the HIV-1 and which can be used as a basis for a potentialvaccine against AIDS.

Rosenberg E. S. et al., Science, Vol. 278, 21 Nov. 1997, p. 1447-1450describe that virus specific CD4+ T helper lymphocytes are critical tothe maintenance of effective immunity in a number of chronic viralinfections, but are characteristically undetectable in chronic humanimmunodeficiency virus-type 1 (HIV-1) infection. HIV-1-specificproliferative responses to p24 were inversely related to viral load.They conclude that the HIV-1-specific helper cells are likely to beimportant in immunotherapeutic interventions and vaccine development.

EP 0 230 222, EP 0 270 114, DE 37 11 016 and GB 2 188 639 all in thename of F. Hoffmann-La Roche & Co. Aktiengesellschaft concernrecombinant expression and purification of an HTLVIII Gag/Env geneprotein or fusionproteins. The proteins consisting of native sequencescan be purified to homogeneity and used as a basis for diagnostic testsfor detection of antibodies against viruses associated with AIDS. Thegag/env protein may also be formulated for use as a vaccine forprotection against AIDS through prophylactic immunization.

International Patent Application WO00/52040 discloses methods fortreating HIV infections by administering e.g. HIV specific peptidesbased on conserved regions of HIV gag p24.

There is a need to provide improved methods for the treatment of HIVinfections and AIDS.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide a moreefficient method for reducing and/or delaying pathological effects ofhuman immunodeficiency virus I (HIV) in a human infected with HIV or forreducing the risk of developing acquired immunodeficiency syndrome(AIDS) in a human, wherein the human potential subject for treatment istested for antibodies against HIV envelope glycoprotein gp120 and/orgp41 to identify subjects having most benefit of a treatment with acomposition, that elicit a cell-mediated immune response in a subject,such as a composition comprising one or more peptide, such as aHIV-specific peptide.

SUMMARY OF THE INVENTION

It has been found that a specific subgroup of human patients infectedwith HIV has an improved response to an HIV vaccine. In particular, itwas found that human patients infected with HIV and which patients hasan amount of antibodies against a defined region of HIV envelopeglycoprotein gp120 and/or gp41 above background level of uninfectedhumans, such as above 0.5 μg/ml, such as above 0.6 μg/ml, such as above0.7 μg/ml, such as above 0.8 μg/ml, such as above 0.9 μg/ml, such asabove 1.0 μg/ml, such as above 1.5 μg/ml, such as above 2 μg/ml, such asabove 3 μg/ml, such as above 4 μg/ml, such as above 5 μg/ml, such asabove 6 μg/ml, such as above 7 μg/ml, such as above 8 μg/ml, such asabove 9 μg/ml, such as above 10 μg/ml of measured antibodiescorresponding to an amount of antibodies against Vacc-C5 in serum asmeasured by an ELISA assay as described in example 33, these patientshas an improved response to a treatment with one or more peptide(s) tostimulate a cell-mediated immune response and/or a compound thatstimulate a humoral response in said human.

It is to be understood that the effect seen in this specific subgroup ofhuman patients infected with HIV is increased not just because thesepatients respond better to the vaccination or are the best patientssuited in general for vaccinations. This is very specific to thevaccination regime according to the present invention.

So, in a first aspect of the present invention is provided a method forreducing and/or delaying pathological effects of human immunodeficiencyvirus I (HIV) or for reducing the risk of developing acquiredimmunodeficiency syndrome (AIDS) in a human infected with HIV, themethod comprising the steps of:

a) measuring in a biological sample, such as serum or plasma, from ahuman infected with HIV the amount of antibodies against one or moreepitope of HIV envelope glycoproteins gp120 and/or gp41 in a suitableassay;

b) selecting a subgroup of humans from a), wherein the amount of saidmeasured antibodies corresponds to an amount of above background level,such as above 1 μg/ml of antibodies against Vacc-C5 in serum as measuredby an ELISA assay as described in example 33;

c) treating said humans infected with HIV selected under b) with one ormore peptide(s) to stimulate a cell-mediated immune response and/or acompound that stimulate a humoral response in said human.

Said measured antibodies corresponding to an amount above background instep b) is understood to mean above 0.5 μg/ml, such as above 0.6 μg/ml,such as above 0.7 μg/ml, such as above 0.8 μg/ml, such as above 0.9μg/ml, such as above 1.0 μg/ml, such as above 1.5 μg/ml, such as above 2μg/ml, such as above 3 μg/ml, such as above 4 μg/ml, such as above 5μg/ml, such as above 6 μg/ml, such as above 7 μg/ml, such as above 8μg/ml, such as above 9 μg/ml, such as above 10 μg/ml of measuredantibodies corresponding to an amount of antibodies against Vacc-C5 inserum as measured by an ELISA assay as described in example 33.

In a second aspect of the present invention is provided a kit forreducing and/or delaying pathological effects of human immunodeficiencyvirus I (HIV) or for reducing the risk of developing acquiredimmunodeficiency syndrome (AIDS) in a human infected with HIV, which kitcomprises

a) a test assay for measuring in a biological sample, such as serum orplasma, of said human infected with HIV the amount of antibodies againstHIV envelope glycoprotein gp120 and/or gp41; and

b) one or more peptide to stimulate a cell-mediated immune responseand/or a compound that stimulate a humoral response in said human,optionally

c) one or more immunomodulatory compound and/or a reservoir purgingagent, such as any one described herein.

In a third aspect of the present invention there is provided aneffective amount of one or more peptide to stimulate a cell-mediatedimmune response and/or a compound that stimulate a humoral response foruse in method for reducing and/or delaying pathological effects of humanimmunodeficiency virus I (HIV) or for reducing the risk of developingacquired immunodeficiency syndrome (AIDS) in a human infected with HIV,the method comprising the steps of:

a) measuring in a biological sample, such as serum or plasma, of saidhuman infected with HIV the amount of antibodies against HIV envelopeglycoprotein gp120 and/or gp41 in a suitable assay;

b) selecting humans infected with HIV, wherein the amount of saidmeasured antibodies corresponds to an amount of above background levelof uninfected humans, such as above 1 μg/ml of antibodies againstVacc-C5 in serum as measured by an ELISA assay as described in example33;

c) treating said humans infected with HIV selected under b) with one ormore peptide to stimulate a cell-mediated immune response and/or acompound that stimulate a humoral response in said human.

In a fourth aspect of the present invention is provided a method forreducing and/or delaying pathological effects of human immunodeficiencyvirus I (HIV) or for reducing the risk of developing acquiredimmunodeficiency syndrome (AIDS) in a human infected with HIV, themethod comprising the steps of:

a) measuring in a biological sample, such as serum or plasma, of saidhuman infected with HIV the amount of antibodies against HIV envelopeglycoprotein gp120 and/or gp41 in a suitable assay;

b) selecting humans infected with HIV, wherein said amount of antibodiesis above background level in uninfected humans and below 1 μg/ml ofantibodies against Vacc-C5 in serum as measured by an ELISA assay asdescribed in example 33;

c) treating said humans infected with HIV selected under b) with acompound that stimulate a humoral response in said human;

d) optionally having a maturation period, such as a period of up to 4weeks;

e) optionally repeating the measurement in a) and selecting humansinfected with HIV, wherein the amount of said measured antibodiescorresponds to an amount of above background level of uninfected humans,such as above 1 μg/ml of antibodies against Vacc-C5 in serum as measuredby an ELISA assay as described in example 33;

f) treating said humans infected with HIV selected under b) or e) withone or more peptide to stimulate a cell-mediated immune response and/ora compound that stimulate a humoral response in said human.

In some embodiments, the method comprises the steps of:

a) measuring in a biological sample, such as serum or plasma, of saidhuman infected with HIV the amount of antibodies against HIV envelopeglycoprotein gp120 and/or gp41 in a suitable assay;

b) selecting humans infected with HIV, wherein said amount of antibodiesis above background level in uninfected humans and below 1 μg/ml ofantibodies against Vacc-C5 in serum as measured by an ELISA assay asdescribed in example 33;

c) treating said humans infected with HIV selected under b) with acompound that stimulate a humoral response in said human;

d) optionally having a maturation period, such as a period of up to 4weeks;

e) treating said humans infected with HIV selected under b) with one ormore peptide to stimulate a cell-mediated immune response and/or acompound that stimulate a humoral response in said human.

In some embodiments, the method comprises the steps of:

a) measuring in a biological sample, such as serum or plasma, of saidhuman infected with HIV the amount of antibodies against HIV envelopeglycoprotein gp120 and/or gp41 in a suitable assay;

b) selecting humans infected with HIV, wherein said amount of antibodiesis above background level in uninfected humans and below 1 μg/ml ofantibodies against Vacc-C5 in serum as measured by an ELISA assay asdescribed in example 33;

c) treating said humans infected with HIV selected under b) with acompound that stimulate a humoral response in said human;

d) having a maturation period, such as a period of up to 4 weeks;

e) repeating the measurement in a) and selecting humans infected withHIV, wherein the amount of said measured antibodies corresponds to anamount of above background level of uninfected humans, such as above 1μg/ml of antibodies against Vacc-C5 in serum as measured by an ELISAassay as described in example 33;

f) treating said humans infected with HIV selected under b) or e) withone or more peptide to stimulate a cell-mediated immune response and/ora compound that stimulate a humoral response in said human.

In some embodiments, the methods according to the present invention ispreceded by a step of treating said humans infected with HIV with acompound that stimulate a humoral response in said human, such as acompound as defined herein.

In some embodiments, the effective amount of one or more peptide tostimulate a cell-mediated immune response and/or a compound thatstimulate a humoral response is for use in a method according to thepresent invention preceded by a step of treating said humans infectedwith HIV with a compound that stimulate a humoral response in saidhuman, such as a compound as defined herein.

It is to be understood that when selecting humans infected with HIV inb) above, the amount of antibodies should preferably be above backgroundlevel of uninfected humans and below 1 μg/ml, such as in the range of0.1 and 0.5 μg/ml, such as in the range of 0.1 μg/ml and 1 μg/ml, suchas in the range of 0.1 and 1.5 μg/ml, such as in the range of 0.1 μg/mland 2 μg/ml, such as in the range of 0.1 and 3 μg/ml, such as in therange of 0.1 and 4 μg/ml, such as in the range of 0.1 and 5 μg/ml, suchas in the range of 0.1 and 6 μg/ml, such as in the range of 0.1 and 7μg/ml, such as in the range of 0.1 and 8 μg/ml, such as in the range of0.1 and 9 μg/ml, such as in the range of 0.1 and 10 μg/ml.

It is also to be understood that after treatment with a compound tostimulate a humoral response in a human a maturation period may beneeded to build up said response, such as a period of two weeks, such asa period of three weeks, such as a period of four weeks, such as aperiod of five weeks, such as a period of six weeks, such as a period ofseven weeks, such as a period of eight weeks, if one i.e. wants tomeasure it.

In a fifth aspect of the present invention there is provided aneffective amount of one or more peptide to stimulate a cell-mediatedimmune response and/or a compound that stimulate a humoral response foruse in method for reducing and/or delaying pathological effects of humanimmunodeficiency virus I (HIV) or for reducing the risk of developingacquired immunodeficiency syndrome (AIDS) in a human infected with HIV,the method comprising the steps of:

a) measuring in a biological sample, such as serum or plasma, of saidhuman infected with HIV the amount of antibodies against HIV envelopeglycoprotein gp120 and/or gp41 in a suitable assay;

b) selecting humans infected with HIV, wherein said amount of antibodiesis above background level in uninfected humans and below 1 μg/ml ofantibodies against Vacc-C5 in serum as measured by an ELISA assay asdescribed in example 33;

c) treating said humans infected with HIV selected under b) with acompound that stimulate a humoral response in said human;

d) optionally having a maturation period, such as a period of up to 4weeks;

e) optionally repeating the measurement in a) and selecting humansinfected with HIV, wherein said amount of antibodies is above 1 μg/ml ofantibodies against Vacc-C5 in serum as measured by an ELISA assay asdescribed in example 33;

f) treating said humans infected with HIV selected under b) or e) withone or more peptide to stimulate a cell-mediated immune response and/ora compound that stimulate a humoral response in said human.

In some embodiments, the method comprises the steps of:

a) measuring in a biological sample, such as serum or plasma, of saidhuman infected with HIV the amount of antibodies against HIV envelopeglycoprotein gp120 and/or gp41 in a suitable assay;

b) selecting humans infected with HIV, wherein said amount of antibodiesis above background level in uninfected humans and below 1 μg/ml ofantibodies against Vacc-C5 in serum as measured by an ELISA assay asdescribed in example 33;

c) treating said humans infected with HIV selected under b) with acompound that stimulate a humoral response in said human;

d) optionally having a maturation period, such as a period of up to 4weeks;

e) treating said humans infected with HIV selected under b) with one ormore peptide to stimulate a cell-mediated immune response and/or acompound that stimulate a humoral response in said human.

In some embodiments, the method comprises the steps of:

a) measuring in a biological sample, such as serum or plasma, of saidhuman infected with HIV the amount of antibodies against HIV envelopeglycoprotein gp120 and/or gp41 in a suitable assay;

b) selecting humans infected with HIV, wherein said amount of antibodiesis above background level in uninfected humans and below 1 μg/ml ofantibodies against Vacc-C5 in serum as measured by an ELISA assay asdescribed in example 33;

c) treating said humans infected with HIV selected under b) with acompound that stimulate a humoral response in said human;

d) having a maturation period, such as a period of up to 4 weeks;

e) repeating the measurement in a) and selecting humans infected withHIV, wherein the amount of said measured antibodies corresponds to anamount of above background level of uninfected humans, such as above 1μg/ml of antibodies against Vacc-C5 in serum as measured by an ELISAassay as described in example 33;

f) treating said humans infected with HIV selected under b) or e) withone or more peptide to stimulate a cell-mediated immune response and/ora compound that stimulate a humoral response in said human.

DETAILED DISCLOSURE OF THE INVENTION

The present invention is based on the finding that human patients,infected with HIV and which patients has an amount of antibodies againsta defined region of HIV envelope glycoprotein gp120 and/or gp41 above 2μg/ml, has an improved response to a HIV vaccine treatment with one ormore peptide to stimulate a cell-mediated immune response and/or acompound that stimulate a humoral response in said human.

DEFINITIONS

The term “biological sample” refers to any body fluid or subfractionthereof that may be obtained from the body of a mammal. Included withinthis definition are cerebrospinal fluids, blood, such as blood from thecirculatory system or from the umbilical cord, serum, lymph fluid,plasma, pleura exudates, peritoneal exudates, bone marrow exudates,extracellular fluids, fluids from the joints, amniotic fluids.

In some embodiments the body fluid is blood, such as peripheral blood orany component derived from blood, such as the serum or plasma fraction.

The phrase “the amount of antibodies against one or more epitope of HIVenvelope glycoproteins gp120 and/or gp41 in a suitable assay” as usedherein refers to the amount of antibodies specifically binding one ormore epitopes present in HIV envelope glycoproteins gp120 and/or gp41,such as those present in Vacc-C5 as defined herein, which may bemeasured in the assay described in example 33. It is to be understoodthat Vacc-C5 is a dimeric peptide consisting of peptides derived fromboth gp120 and gp41 and accordingly comprising epitopes derivedtherefrom. The specific amounts may vary somewhat depending on thespecific assay used. It is also to be understood that a humoral immuneresponse to one or more epitope of HIV envelope glycoproteins gp120and/or gp41 may be measured with other antigens derived from gp120and/or gp41 and that Vacc-C5 is just one measure for antibodies againstone or more epitope of HIV envelope glycoproteins gp120 and/or gp41. Thethreshold values are set corresponding to values obtained when usingantibodies against Vacc-C5 in serum as a standard and when using thespecific assay described herein. This does not exclude that other assaysmay be used or that other specific epitopes derived from HIV envelopeglycoproteins gp120 and/or gp41 may be used to detect an elevated levelof antibodies against one or more epitope of HIV envelope glycoproteinsgp120 and/or gp41.

When terms such as “one”, “a” or “an” are used in this disclosure theymean “at least one”, or “one or more” unless otherwise indicated.Further, the term “comprising” is intended to mean “including” and thusallows for the presence of other constituents, features, conditions, orsteps than those explicitly recited.

“HIV” unless otherwise indicated generally denotes humanimmunodeficiency virus I.

“HIV disease” is composed of several stages including the acute HIVinfection which often manifests itself as a flu-like infection and theearly and medium stage symptomatic disease, which has severalnon-characteristic symptoms such as skin rashes, fatigue, night sweats,slight weight loss, mouth ulcers, and fungal skin and nail infections.Most HIV infected will experience mild symptoms such as these beforedeveloping more serious illnesses. It is generally believed that ittakes five to seven years for the first mild symptoms to appear. As HIVdisease progresses, some individuals may become quite ill even if theyhave not yet been diagnosed with AIDS (see below), the late stage of HIVdisease. Typical problems include chronic oral or vaginal thrush (afungal rash or spots), recurrent herpes blisters on the mouth (coldsores) or genitals, ongoing fevers, persistent diarrhea, and significantweight loss. “AIDS” is the late stage HIV disease and is a conditionwhich progressively reduces the effectiveness of the immune system andleaves individuals susceptible to opportunistic infections and tumors.

When using the term “gp120” herein is meant the ≈120 kDa N-terminalglycoprotein enzymatic cleavage product of gp160, which in turn is thesole expression product of the HIV env gene. gp120 forms the “spikes” oninfective HIV virions and is non-covalently bound to gp41.

“gp41” denotes the ≈41 kDa glycoprotein C-terminal enzymatic cleavageproduct of gp160. gp41 is located in the membrane of HIV infected cellsor virions. gp41 has an N-terminal transmembrane domain which bindsnon-covalently to gp120. This transmembrane domain is termed “thetransmembrane domain of gp41” or “tm-gp41” herein. The term includeswithin its scope naturally occurring mutated versions of the sequence ase.g. those set forth in Formula III.

“C5” or the “C5 domain” denotes the 13 C-terminal amino acid residues ofgp120.

“C2” or the “C2 domain” denotes a conserved region in gp120. Regions inC2 form an antiparallel β-sheet with C5 in the inner proximal domain ofgp12.

“Reducing and/or delaying pathological effect of HIV” is in the presentcontext meant to denote that use of the methods of the inventionprovides for a statistically significant reduction and/or delay inmorbidity seen in individual infected with HIV which are treatedaccording to the present invention. That is, the time of onset ofmanifest disease symptoms characterizing AIDS is later compared tonon-treated controls and/or the number of pathological manifestations isreduced to controls not receiving the treatment of the presentinvention.

The expression “association of the C5 domain of HIV gp120 with thetransmembrane domain of gp41 and/or with the constant C2 domain ofgp120” means that C5 can interact non-covalently with both or one of thetm-g41 and C2. The interaction with tm-gp41 is intermolecular, whereasthe interaction with C2 is intramolecular.

An “agent capable of stabilising” association of the C5 domain of HIVgp120 with the transmembrane domain of gp41 and/or with the constant C2domain of gp120 is a composition of matter which prevents orstatistically reduces release of C5 from its intermolecular binding togp41 and/or from its intramolecular binding to C2. Generally, such anagent is any substance of matter capable of exerting this effect, butimportant examples are antibodies, antibody fragments, and antibodyanalogues. However, also other molecules having proper binding affinityfor a complex between C5 on the one hand and tm-gp41 and/or C2 on theother, is an agent according the present invention—the precise molecularformat is less important than the binding characteristics, and it isaccording to the invention also possible that such an agent may be areceptor or a receptor analogue, but also small molecule stabilisers arecapable of functioning as an agent of the present invention.

The term “antibody” herein is used in the broadest sense andspecifically includes full-length monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments, so long as they exhibit the desired biologicalactivity, i.e. to function as an agent described above. Varioustechniques relevant to the production of antibodies are provided in,e.g., Harlow, et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).).

An “antibody fragment or antibody analogue” comprises a portion of afull-length antibody, preferably antigen-binding or variable regionsthereof. Examples of antibody fragments/analogues include Fab, Fab′,F(ab)₂, F(ab′)₂, F(ab)₃, Fv (typically the VL and VH domains of a singlearm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments(typically the VH and CH1 domain), and dAb (typically a VH domain)fragments; VH, VL, VhH, and V-NAR domains; minibodies, diabodies,triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al.,Protein Eng 1997; 10: 949-57); camel IgG; IgNAR; and multispecificantibody fragments formed from antibody fragments, and one or moreisolated CDRs or a functional paratope, where isolated CDRs orantigen-binding residues or polypeptides can be associated or linkedtogether so as to form a functional antibody fragment. Various types ofantibody fragments have been described or reviewed in, e.g., Holligerand Hudson, Nat Biotechnol 2005; 23, 1126-1136; WO2005040219, andpublished U.S. Patent Applications 20050238646 and 20020161201.

The term “antibody derivative”, as used herein, comprises a full-lengthantibody or a fragment of an antibody, preferably comprising at leastantigen-binding or variable regions thereof, wherein one or more of theamino acids are chemically modified, e.g., by alkylation, PEGylation,acylation, ester formation or amide formation or the like, e.g., forlinking the antibody to a second molecule. This includes, but is notlimited to, PEGylated antibodies, cysteine-PEGylated antibodies, andvariants thereof.

A “conjugate” as used herein comprises an agent to be used according tothe invention such as an antibody derivative associated with or linkedto a second agent, such as a cytotoxic agent, a detectable agent, etc. Aconjugate may be constituted of covalently linked peptides (an exampleof a conjugate is a fusion peptide comprising two peptides linked viapeptide bonds so that the conjugate in that case may be an expressionproduct from a nucleic acid fragment), but a conjugate can also be acombination of peptides covalent linked via chemical conjugation (atraditional example is conjugation using glutaraldehyde). Anotherexample of a more complex conjugation is the example where an agent orpeptide multimer or other chemical substance of the present invention islinked to a carrier molecule, which in turn i coupled to other agents,peptide multimers or other chemical substances of the present invention(e.g. when such chemical substances are bound to a poly-lysine carrier(a lysine “tree”)).

A “humanized” antibody is a human/non-human chimeric antibody thatcontains a minimal sequence derived from non-human immunoglobulin. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a hypervariable region of the recipientare replaced by residues from a hypervariable region of a non-humanspecies (donor antibody) such as mouse, rat, rabbit, or non-humanprimate having the desired specificity, affinity, and capacity. In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are not found in the recipientantibody or in the donor antibody. These modifications are made tofurther refine antibody performance. In general, a humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableloops correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinsequence. The humanized antibody can optionally also comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details, see, e.g., Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), WO 92/02190, USPatent Application 20060073137, and U.S. Pat. Nos. 6,750,325, 6,632,927,6,639,055, 6,548,640, 6,407,213, 6,180,370, 6,054,297, 5,929,212,5,895,205, 5,886,152, 5,877,293, 5,869,619, 5,821,337, 5,821,123,5,770,196, 5,777,085, 5,766,886, 5,714,350, 5,693,762, 5,693,761,5,530,101, 5,585,089, and 5,225,539.

An antibody having a “biological characteristic” of a referenceantibody, is one that possesses one or more of the biologicalcharacteristics of that antibody that distinguish it from otherantibodies that bind to the same antigen.

The term “peptide” is in the present context intended to mean both shortpeptides of from 2 to 10 amino acid residues, oligopeptides of from 11to 100 amino acid residues, and polypeptides of more than 100 amino acidresidues. When referring to amino acids in peptides, it is intended thatthe amino acids are L-amino acids, unless other information is provided.Amino acids are referred to by their standard three letter or one letterdesignations unless otherwise stated. Some unusual amino acids referredto herein includes homoarginine usually abbreviated by Har, norleucineusually abbreviated as Nle or NI, N-ε-methylated Lys usually abbreviatedLys(Me), Citrulline usually abbreviated Cit or with the single letter“B”, diaminopropionic acid usually abbreviated with Dpr and serinyldiaminopropionic acid usually abbreviated Dpr(Ser).

A “protein” is intended to denote a functional biomolecule comprising atleast one peptide; when comprising at least two peptides, these may formcomplexes, be covalently linked, or may be non-covalently linked. Thepolypeptide(s) in a protein can be glycosylated and/or lipidated and/orcomprise prosthetic groups.

A “peptide multimer” denotes a molecule which is constituted by at leasttwo peptides in a non-natural configuration relative to each other.Examples are peptides from the same or from different proteins which arecovalently linked via the side chains of at least one of their aminoacids, or which are linked via their termini (e.g. via peptide bonds)but in a configuration which does not appear in nature. Typical examplesof peptide multimers are detailed below.

A “variant” or “analogue” of a peptide refers to a peptide having anamino acid sequence that is substantially identical to a referencepeptide, typically a native or “parent” polypeptide. The peptide variantmay possess one or more amino acid substitutions, deletions, and/orinsertions at certain positions within the native amino acid sequence.

“Conservative” amino acid substitutions are those in which an amino acidresidue is replaced with an amino acid residue having a side chain withsimilar physicochemical properties. Families of amino acid residueshaving similar side chains are known in the art, and include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Aparticular form of conservative amino acid substitutions include thosewith amino acids, which are not among the normal 20 amino acids encodedby the genetic code. Since preferred embodiments of the presentinvention entail use of synthetic peptides, it is unproblematic toprovide such “non-naturally occurring” amino acid residues in thepeptides disclosed herein, and thereby it is possible to exchange thenatural saturated carbon chains in the side chains of amino acidresidues with shorter or longer saturated carbon chains—for instance,lysine may be substituted with an amino acid having an the side chain—(CH₂)_(n)NH₃, where n is different from 4, and arginine may besubstituted with an amino acid having the side chain—(CH₂)_(n)NHC(═NH₂)NH₂, where n is different from 3, etc. Similarly, theacidic amino acids aspartic acid and glutamic acid may be substitutedwith amino acid residues having the side chains —(CH₂)_(n)COOH, wheren>2.

A “retro form” of a peptide is a form of a peptide where the order ofthe amino acids in N- to C-terminal direction has been inverted. Forinstance, the retro form of ALDFR is the peptide RFDLA.

The term “substantially identical” in the context of two amino acidsequences means that the sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at leastabout 50, at least about 60, at least about 70, at least about 80, atleast about 90, at least about 95, at least about 98, or at least about99 percent sequence identity. In one embodiment, residue positions thatare not identical differ by conservative amino acid substitutions.Sequence identity is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, the publicly available GCG software contains programs suchas “Gap” and “BestFit” which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild-type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences can also be compared usingFASTA or ClustalW, applying default or recommended parameters. A programin GCG Version 6.1., FASTA (e.g., FASTA2 and FASTA3) provides alignmentsand percent sequence identity of the regions of the best overlap betweenthe query and search sequences (Pearson, Methods Enzymol. 1990;183:63-98; Pearson, Methods Mol. Biol. 2000; 132:185-219). Anotherpreferred algorithm when comparing a sequence to a database containing alarge number of sequences from various organisms, or when deducing theis the computer program BLAST, especially blastp, using defaultparameters. See, e.g., Altschul et al., 3. Mol. Biol. 1990; 215:403-410;Altschul et al., Nucleic Acids Res. 1997; 25:3389-402 (1997); eachherein incorporated by reference. “Corresponding” amino acid positionsin two substantially identical amino acid sequences are those aligned byany of the protein analysis software mentioned herein, typically usingdefault parameters.

The term “subsequence” in general means any consecutive stretch of atleast 3 amino acids or, when relevant, of at least 3 nucleotides,derived directly from a naturally occurring amino acid sequence ornucleic acid sequence, respectively. However, when discussing peptidemultimers of the present invention, the subsequence may be as short as 1or 2 amino acids. This is because the inventive peptide multimersinclude amino acids from different peptide domains, where the aminoacids together at least form a conformational epitope for an antibody.Hence, such a conformational epitope could be composed of 4 amino acidsfrom C5, but only 1 or 2 from tm-gp41—the important point is here thatthis combined epitope from 2 domains is capable of being stabilised,i.e. that antibody binding to the same epitope in vivo will stabilisethe configuration between C5 and tm-gp41 and/or C2.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome-binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

An “isolated” molecule is a molecule that is the predominant species inthe composition wherein it is found with respect to the class ofmolecules to which it belongs (i.e., it makes up at least about 50% ofthe type of molecule in the composition and typically will make up atleast about 70%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more of the species of molecule, e.g.,peptide, in the composition). Commonly, a composition of an antibodymolecule will exhibit 98%-99% homogeneity for antibody molecules in thecontext of all present peptide species in the composition or at leastwith respect to substantially active peptide species in the context ofproposed use.

In the context of the present invention, “treatment” or “treating”refers to preventing, alleviating, managing, curing or reducing one ormore symptoms or clinically relevant manifestations of a disease ordisorder, unless contradicted by context. For example, “treatment” of apatient in whom no symptoms or clinically relevant manifestations of adisease or disorder have been identified is preventive or prophylactictherapy, whereas “treatment” of a patient in whom symptoms or clinicallyrelevant manifestations of a disease or disorder have been identifiedgenerally does not constitute preventive or prophylactic therapy.

The term antigen denotes a substance of matter which is recognized bythe immune system's specifically recognizing components (antibodies,T-cells).

The term “immunogen” is in the present context intended to denote asubstance of matter, which is capable of inducing an adaptive immuneresponse in an individual, where said adaptive immune response targetsthe immunogen. In other words, an immunogen is an antigen, which iscapable of inducing immunity.

The terms “epitope”, “antigenic determinant” and “antigenic site” areused interchangeably herein and denotes the region in an antigen orimmunogen which is recognized by antibodies (in the case of antibodybinding epitopes, also known as “B-cell epitopes”) or by T-cellreceptors when the epitope is complexed to an MHC molecule (in the caseof T-cell receptor binding epitopes, i.e. “T-cell epitopes”).

The term “immunogenically effective amount” has its usual meaning in theart, i.e. an amount of an immunogen, which is capable of inducing animmune response, which significantly engages pathogenic agents, whichshare immunological features with the immunogen.

The term “vaccine” is used for a composition comprising an immunogen andwhich is capable of inducing an immune response which is either capableof reducing the risk of developing a pathological condition or capableof inducing a therapeutically effective immune response which may aid inthe cure of (or at least alleviate the symptoms of) a pathologicalcondition.

The term “pharmaceutically acceptable” has its usual meaning in the art,i.e. it is used for a substance that can be accepted as part of amedicament for human use when treating the disease in question and thusthe term effectively excludes the use of highly toxic substances thatwould worsen rather than improve the treated subject's condition.

A “T helper lymphocyte epitope” (a T_(H) epitope) is peptide, whichbinds an MHC Class II molecule and can be presented on the surface of anantigen presenting cell (APC) bound to the MHC Class II molecule. An“immunological carrier” is generally a substance of matter whichincludes one or many T_(H) epitopes, and which increase the immuneresponse against an antigen to which it is coupled by ensuring thatT-helper lymphocytes are activated and proliferate. Examples of knownimmunological carriers are the tetanus and diphtheria toxoids andkeyhole limpet hemocyanin (KLH).

The term “adjuvant” has its usual meaning in the art of vaccinetechnology, i.e. a substance or a composition of matter which is 1) notin itself capable of mounting a specific immune response against theimmunogen of the vaccine, but which is 2) nevertheless capable ofenhancing the immune response against the immunogen. Or, in other words,vaccination with the adjuvant alone does not provide an immune responseagainst the immunogen, vaccination with the immunogen may or may notgive rise to an immune response against the immunogen, but the combinedvaccination with immunogen and adjuvant induces an immune responseagainst the immunogen which is stronger than that induced by theimmunogen alone.

Peptides that Stimulate Cell-Mediated Immunity (CMI)

Vaccination aims to stimulate the immune response to a specific pathogenin advance of infection. When an individual is exposed to that pathogen,a memory response is triggered which prevents the establishment ofinfection. Vaccines therefore stimulate the adaptive immune responsewhich unlike innate immunity, is long lived and has memory.

There are two major arms to the adaptive immune system. Humoral immunitywhich involves the development of antibodies that can bind virusparticles and certain antibodies that can neutralize infection. Cellmediated immunity that leads to the development of cytotoxic T-cellsthat kill infected cells exposing viral epitopes in the context of humanleukocyte antigen (HLA) class I, in this way eliminating infected cells.

As used herein a peptide that elicits a cell-mediated immune responserefers to any peptide that elicits an activation of antigen-specificcytotoxic T-lymphocytes. These peptides elicit a Cytotoxic T-lymphocyteimmune (CTL) response that leads to the development of cytotoxic T-cellsthat kill infected cells exposing viral epitopes in the context of humanleukocyte antigen (HLA) class I, in this way eliminating infected cells.Cell-mediated immunity (CMI) may also involve the activation ofphagocytes, natural killer cells (NK), antigen-specific cytotoxicT-lymphocytes, and the release of various cytokines in response to anantigen.

These peptides may be a helper T lymphocyte (HTL) inducing peptidecomprising HTL epitopes. A “HTL inducing peptide” is a HLA Class IIbinding peptide that is capable of inducing a HTL response. Also thepeptides may in other embodiments be CTL inducing peptides comprisingCTL epitopes in addition to or as an alternative to being a HTL inducingpeptide. A “CTL inducing peptide” is a HLA Class I binding peptide thatis capable of inducing a CTL response.

A peptide that elicit a cell-mediated immune response as used accordingto the present invention includes but is not limited to any peptidedescribed in any one of international patent applications WO0052040, WO2012/092934 or WO 2012/072088, which patent applications are herebyincorporated by reference.

HIV-Specific Peptides

In some aspects, the compositions according to the present inventioncomprise one or more peptide that elicits a cell-mediated immuneresponse. In some embodiments, this peptide is at least one HIV-specificpeptide.

One aspect of the peptides that elicits a cell-mediated immune responserelates to HIV-specific peptides based on conserved regions of HIV gagp24, antigens in free or carrier-bound form comprising at least one ofthe said peptides.

The HIV-specific peptides to be used according to the invention mayoriginate from conserved areas of the HIV-1 core protein p24, having theproperties of maintaining the uniqueness (sensitivity and specificity)of the HIV-1-epitope. Further the new peptides to be used according tothe invention possess no recognized cytotoxic T lymphocyte (CTL)antagonistic effect and shall have at least one potential CTL epitope.

In some embodiments, the HIV-specific peptides, to be used according tothe present invention, and which have met the above criteria areselected from the following groups;

(SEQ ID NO: 47) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Ala Xaa₈ Xaa₉ Gln Thr ProTrp Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Val Xaa₂₀;Wherein Xaa in position 1 of the peptide derivate is Lys or Arg,Xaa in position 2 is Ala, Gly, Ser or Arg,Xaa in position 3 is Leu or Met,Xaa in position 4 is Gly or Arg,Xaa in position 5 is Pro, Thr, Val, Ser, Gln or Ala,Xaa in position 6 is Gly, Ala, Lys, Arg, Gln or Glu,Xaa in position 8 is Thr or Ser,Xaa in position 9 is Leu or Ile,Xaa in position 14 is Thr, Ser or Val,Xaa in position 15 is Ala or Ser,Xaa in position 16 is Cys or Ser,Xaa in position 17 is Gln or LeuXaa in position 18 is Gly, Glu or Arg, andXaa in position 20 is Gly or Arg;

(SEQ ID NO: 50) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Gly Leu Asn Pro Leu Val[Gly]_(n) Xaa₁₂ Xaa₁₃ Tyr Xaa₁₅ Pro Xaa₁₇ Xaa₁₈ Ile Leu Xaa₂₁ Xaa₂₂wherein Xaa in position 1 is Arg, Lys, Asp or noneXaa in position 2 is Trp, Gly, Lys or Arg,Xaa in position 3 is Ile, Leu, Val or MetXaa in position 4 is Ile, Val or LeuXaa in position 5 Leu, Met, Val or ProXaa in position 12 is Arg, LysXaa in position 13 is Met or Leu,Xaa in position 15 is Ser, Cys or Gln,Xaa in position 17 is Thr, Val, Ile, Ser or Ala,Xaa in position 18 is Ser, Gly or Thr,Xaa in position 21 is Asp, Glu, Cys or Gly,Xaa in position 22 is Gly or none, andn=0, 1, 2 or 3;

(SEQ ID NO: 55) Xaa₁ Xaa₂ Xaa₃ Pro Ile Pro Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁Xaa₁₂ [Gly]_(n) Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁Xaa₂₂ Xaa₂₃ Xaa₂₄wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or noneXaa in position 2 is Asn, Ala or LysXaa in position 3 is Pro, Gln, Gly, Ile or LeuXaa in position 7 is Val or AlaXaa in position 8 is Gly or LysXaa in position 9 is Glu, Asp, Lys, Phe or ThrXaa in position 10 is Ile, Met, Val or LeuXaa in position 11 is Tyr, Leu or noneXaa in position 12 is Ser or noneXaa in position 13 is Arg or noneXaa in position 14 is Asp, Arg, Trp, Ala or noneXaa in position 15 is Ile or noneXaa in position 16 is Tyr or noneXaa in position 17 is Lys or ArgXaa in position 18 is Arg, Lys or AspXaa in position 19 is Trp or GlyXaa in position 20 is Ile, Met, Val, Gln or AlaXaa in position 21 is Ile, Val or AlaXaa in position 22 is Leu, Met or ValXaa in position 23 is Gly or CysXaa in position 24 is Leu or none,n=1, 2 or 3, and

(SEQ ID NO: 61) Xaa₁ Xaa₂ Ile Ile Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Leu Xaa₁₁[Gly]_(n) [Arg]_(m) Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅wherein the Xaa in position 1 is Pro, Lys, Arg or noneXaa in position 2 is Glu, Arg, Phe or LysXaa in position 5 is Pro or ThrXaa in position 6 is Met, Thr or NleuXaa in position 7 is Phe or LeuXaa in position 8 is Ser, Thr, Ala or MetXaa in position 9 is Ala, Glu or LeuXaa in position 11 is Ser or noneXaa in position 12 is Ala, Arg or noneXaa in position 13 is Ile, Leu or noneXaa in position 14 is Ser, Ala, Leu or noneXaa in position 15 is Tyr, Glu or AspXaa in position 16 is Gly or AspXaa in position 17 is Ala or LeuXaa in position 18 is Thr, Ile, Val, Leu or Asn,Xaa in position 19 is Pro, Thr or SerXaa in position 20 is Tyr, Phe, Nleu, His or GlnXaa in position 21 is Asp, Asn, Leu or AlaXaa in position 22 is Leu, Ile, Val or AsnXaa in position 23 is Asn, Tyr, Cys or GlyXaa in position 24 is Thr, Met, Ile, Ala, Val or noneXaa in position 25 is Gly or nonen=1, 2 or 3 and m=0, 1, 2 or 3 independent of each other,the terminal ends of each HIV specific peptide may be free carboxyl- oramino groups, amides, acyls, acetyls or salts thereof.

The HIV-specific peptide sequences have the potential to serve as a goodantigen wherein the antigen comprises at least one peptide selected fromthe group of sequences of SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55 orSEQ ID NO:61. The antigenicity may be adapted through adjusting theratio or concentration of different peptides or size of the peptides byfor instance dimerisation or polymerisation and/or immobilisation to asolid phase. The antigen comprises two or more polypeptide sequences, tobe used according to the invention, which are either linked by a bridgefor instance a disulphide bridge between the Cys residues of the chainsor bridges like C₁-C₈alkylen possibly intervened by one or moreheteroatoms like O, S, or N or preferably they are unlinked. The chainsmay be immobilized to a solid phase in monomeric, dimeric or oligomericforms. Further amino acids may be added to the ends in order to achievean <<arm>> to facilitate immobilization.

All amino acids in the HIV-specific peptides to be used according to theinvention can be in both D- or L-form, although the naturally occurringL-form is preferred.

The C- and N-terminal ends of the HIV-specific peptide sequences coulddeviate from the natural sequences by modification of the terminalNH₂-group and/or COOH-group, they may for instance be acylated,acetylated, amidated or modified to provide a binding site for a carrieror another molecule.

The HIV-specific peptides to be used according to the invention areconsisting of 6 to 50 amino acids, preferably between 10 and 30 aminoacids. They are covering all natural variation of amino acids in theidentified positions.

The polypeptide antigen to be used according to the invention is eitherin a free or in a carrier-bound form. The carrier or solid phase towhich the peptide is optionally bound can be selected from a widevariety of known carriers. It should be selected with regard to theintended use of the immobilized polypeptide as a diagnostic antigen oras an immunizing component in a vaccine.

Examples of carriers that can be used for e.g. diagnostic purposes aremagnetic beads or latex of co-polymers such as styrene-divinyl benzene,hydroxylated styrene-divinyl benzene, polystyrene, carboxylatedpolystyrene, beads of carbon black, non-activated or polystyrene orpolyvinyl chloride activated glass, epoxy-activated porous magneticglass, gelatine or polysaccharide particles or other protein particles,red blood cells, mono- or polyclonal antibodies or fab fragments of suchantibodies.

In one embodiment of the vaccine according to the present inventioncomprises antigens containing the peptides of the SEQ ID NO:47, 50, 55and 61, more preferred the peptides occur in the ratio 1:1:1:1.

In a further preferred embodiment the vaccine composition contains theantigens;

(SEQ ID NO: 49) RALGPAATLQTPWTASLGVG-NH₂ (SEQ ID NO: 52)RWLLLGLNPLVGGGRLYSPTSILG-NH₂ (SEQ ID NO: 57)RAIPIPAGTLLSGGGRAIYKRTAILG-NH₂ and (SEQ ID NO: 64)RFIIPNIFTALSGGRRALLYGATPYAIG-NH₂. (NI in position 6 is Norleucine).

One of the sequences contains a B-cell epitope and will activate thehumoral immune system, whereas the other sequences contribute withCTL-epitopes and the amino acid changes implemented within the frame ofthe CTL-epitope are designed to achieve enhanced binding. Other aminoacid changes have been conducted in order to facilitate the synthesis ofthe peptide and/or increase the solubility of the peptide.

Compound that Stimulate a Humoral Response in a Subject, Such as C5Related Compounds:

The present invention also relates to the use of compounds thatstimulate the humoral immunity in a subject. Humoral immunity involvesthe development of antibodies that can bind virus particles and certainantibodies that can neutralize infection.

A peptide that stimulate the humoral immunity in a subject as usedaccording to the present invention includes but is not limited to anypeptide described in any one of international patent applicationsWO2011/000962, WO0052040, WO 2012/092934 or WO 2012/072088, which patentapplications are hereby incorporated by reference.

In some embodiments these compounds are agents capable of stabilisingthe association of the C5 domain of HIV gp120 with the transmembranedomain of gp41 and/or with the constant C2 domain of gp120. In otherembodiments these compounds are immunogens, which induces antibodiesthat stabilise association of the C5 domain of HIV gp120 with thetransmembrane domain of gp41 and/or with the constant C2 domain ofgp120.

One aspect of the invention relates to compositions and method forreducing and/or delaying pathological effects of human retrovirusinfection, such as immunodeficiency virus I (HIV) in a human infectedwith such virus, such as HIV, the method including administering aneffective amount of an agent capable stabilising association of the C5domain of HIV gp120 with the transmembrane domain of gp41 and/or withthe constant C2 domain of gp120.

Another aspect is much similar, but relates to compositions and methodsof reducing the risk of developing acquired immunodeficiency syndrome(AIDS), the method including administering an effective amount of anagent capable of stabilising association of the C5 domain of HIV gp120with the transmembrane domain of gp41 and/or with the constant C2 domainof gp120.

These aspects primarily aim at treating retrovirus infections, such asHIV infected individuals with agents, which can mimic the antibodieswhich according to the present invention are characteristic for HIVinfected long-term non-progressors—this is the most straightforwardtherapeutic utilisation of the findings underlying the presentinvention. Where the one aspect aims at reducing pathological effects ofretrovirus infections, such as HIV or prolonging the time it takes todevelop manifest AIDS, the other aspect aims at reducing the risk ofdeveloping AIDS altogether and may therefore be used in individualswhich are currently treated prophylactically with antiretroviraltherapy.

In one embodiment, the agent in these first aspects of the invention isa molecule comprising at least one amino acid sequence selectedindependently from an amino acid sequence derived from the transmembranedomain of gp41 and an amino acid sequence derived from the C2 domain,wherein the at least one amino acid sequence binds the C5 domain andoptionally comprises at least one D-amino acid; in certain embodimentsall the amino acids in the amino acid sequence are D-amino acids. Themolecule is preferably a peptide, and in certain embodiments thispeptide consists of the at least one amino acid sequence. The amino acidsequences typically include at most 10 amino acid residues, such as atmost 9, at most 8, at most 7, at most 6, and at most 5 amino acidresidues. Preferred molecules are therefore peptides having 4, 5, 6, 7,8, 9, or 10 amino acid residues. Specific embodiments of the at leastone molecule are therefore the peptides having or comprising SEQ IDNO:34, 35, 36, 37, 39, 40, 42, 43 and 45, which may all be composedpartly or entirely of D-amino acids. Also molecules comprising peptideshaving Formula III are interesting embodiments of the at least onemolecule.

In one embodiment, the agent in these first aspects of the invention isselected from an antibody, an antibody fragment or an antibody analogue.The antibody may be a fully human antibody, a humanized antibody, or achimeric antibody, or a derivative thereof. Typically, the antibody isan IgA, an IgD, an IgG, an IgE or an IgM—the antibody may be bothmonoclonal and polyclonal. The antibody fragment is typically selectedfrom a Fab fragment, a Fab′ fragment, a Fab′-SH fragment, a F(ab)2fragment, a F(ab′)2 fragment, an Fv fragment, a Heavy chain Ig (a llamaor camel Ig), a V_(HH) fragment, a single domain FV, and a single-chainantibody fragment, and the antibody analogue is typically selected froma scFV, a dsFV, a minibody, a diabody, a triabody, a kappa body, anIgNAR, a tandAb, a BITE, and a multispecific antibody.

In one embodiment of these first aspects of the invention, the agentbinds to and stabilises association between one or more amino acidresidues in the amino acid stretch TZ¹AKRRVVZ²REKR, where Z¹ is K, R orE and where Z² is Q or E, and one or more amino acid residues in anamino acid stretch in the transmembrane domain of gp41 and/or in theconstant C2 domain of gp120. This amino acid stretch from C5 is highlyconserved across the multiple HIV clades known and effective interactionwith this stretch by the agent is therefore believed to be highlyadvantageous.

A further aspect of the invention relates to a method for reducing therisk of or reducing and/or delaying pathological effects of humanimmunodeficiency virus I (HIV) in a human infected with HIV, the methodincluding administering an effective amount of an immunogen, whichinduces antibodies that stabilise association of the C5 domain of HIVgp120 with the transmembrane domain of gp41 and/or with the constant C2domain of gp120, whereas other aspects relates to a prophylactic methodusing the same means. In other words, one aspect relates to therapeuticactive immunotherapy, whereas another aspect relates to prophylacticimmunotherapy of HIV disease, including AIDS. This also entailsprophylaxis of HIV infection.

These particular aspects are based on the realisation that it isfeasible to induce the same type of antibody repertoire in the averageHIV infected individual as the one that is found in the HIV LTNPindividuals. By carefully selecting peptide regions in both C5 and intm-gp41 and/or C2 in order to prepare peptide multimers that mimic theantibody binding epitopes present in HIV composed of these regions, itbecomes possible to prepare vaccines which will induce the desiredimmunity—interestingly, this approach does not aim at vaccinating so asto obtain neutralizing antibodies in the classical sense.

In one embodiment the immunogen is selected from a peptide multimerdetailed below when discussing these aspects of the invention, acomposition detailed below, a nucleic acid fragment discussed inrelation to other aspects, a virus or plasmid vector compositionsdiscussed elsewhere.

In common for the first aspects is that they all include embodimentswhere the targeted association between the C5 domain and C2 and/or thetransmembrane domain of gp41 involves at least one amino acid in thesequence TZ¹AKRRVVZ²REKR, where Z¹ is K, R or E and where Z² is Q or Eand an amino acid and involves at least one amino acid in thetransmembrane domain of gp41 or at least one amino acid in the constantC2 domain of gp120. As explained above, this particular sequence isextremely well-conserved across known HIV clades, and therefore it isthe interaction between this sequence and tm-gp41 or C2 it is mostfeasible to target.

Another aspect relates to a composition comprising (1) a peptidemultimer, said multimer comprising

-   -   a first peptide comprising the amino acid sequence of the 13        amino acid residue amino acid sequence of the C5 domain of HIV        gp120 including between 0 and 4 amino acid substitutions, a        subsequence thereof, or an amino acid sequence comprising the        inverso-, retro- or retro-inverso form of said amino acid        sequence or subsequence, and    -   at least one second peptide comprising an amino acid stretch        present in the transmembrane domain of gp41 or present in the        constant C2 domain of gp120 or comprising an amino acid stretch        present in any one of SEQ ID NOs. 6-13 or comprising an        inverso-, retro- or retro-inverso form of an amino acid stretch        present in the transmembrane domain of gp41 or present in the        constant C2 domain of gp120,

wherein said peptide multimer is capable of inducing an antibody whichcan bind and stabilise the association of the C5 domain of HIV gp120with the transmembrane domain of gp41 and/or with the constant C2 domainof gp120, and wherein said peptide multimer lacks amino acids N-terminalof C5 in gp120.

In other words, this aspect relates to peptide multimers which have aresemblance in 3 dimensions with the epitopes which characterise theinteracting areas in C5 on the one hand and tm-gp41 and/or C2 on theother. The peptide multimers to be used according to the invention areuseful immunogens that can induce antibodies having the samecharacteristics as the antibodies found in HIV LTNP individuals, but thepeptide multimers also are promising as diagnostic/prognostic tools. Theinclusion of retro-, inverso-, and retro-inverso peptides i.a. enablesproduction of proteolytically stable peptides as well as peptides aretruly foreign compared to the HIV counterpart.

In one embodiment of the peptide multimer, said first peptide comprisesthe amino acid sequence having formula I:

X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³  (I)

wherein X¹ is Thr, X² is selected from Lys, Arg, Har and Glu, X³ isselected from Ala and Val,X⁴ is selected from Arg, Har, Lys and Cit (citrulline), X⁵ is selectedfrom Arg, Har, Lys and Cit,X⁶ is selected from Arg, Har, Lys and Cit, X⁷ is selected from Val, Leu,Ile and Nle (norleucin),X⁸ is selected from Val, Leu Ile and Nle, X⁹ is selected from Gln, Glu,Asn and Asp, X¹⁰ is selected from Arg, Har and Cit, X¹¹ is selected fromGlu and Asp, X¹² is Lys, and X¹³ is selected from Arg, Har and Cit,or comprises a subsequence the amino acid sequence of formula I, orcomprising the inverso-, retro- or retro-inverso form of said amino acidsequence or subsequence. The first peptide may further comprise thedipeptide Ala-Pro linked to the N-terminus of the amino acid sequencehaving formula I and/or the first peptide may further comprise thedipeptide X¹⁴⁻X¹⁵ linked to the C-terminus of the amino acid sequencehaving formula I, wherein X¹⁴ is selected from Ala and Val, and whereinX¹⁵ is selected from Val, Leu and Nle.Particularly interesting peptides derived from C5 are set forth in thepreamble to the Examples and constitute embodiments of a first peptideof the peptide multimers to be used according to the invention.

A number of naturally occurring mutants of gp41 and gp120 has beenobserved, so when stating that the second peptide comprises an aminoacid stretch present in the transmembrane domain of gp41 or present inthe constant C2 domain of gp120, this is intended to denote that theamino acid stretch is present in any such naturally occurring form. So,the at least second peptide, when derived from gp41, is in certainembodiments one which includes the amino acid sequence having theformula:

Z¹-Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-Z¹¹-Z¹²-Z¹³-Z¹⁴-Z¹⁵-Z¹⁶-Z¹⁷  (III)

-   -   wherein Z¹ is Asp, Z² is Arg, Z³ is Pro, Z⁴ is Glu or Gly, Z⁵ is        Gly or Arg, Z⁶ is Ile, Z⁷ is Glu, Z⁸ is Glu, Z⁹ is Glu, Z¹⁰ is        Gly, Z¹¹ is Gly, Z¹² is Glu or is absent, Z¹³ is Arg or Gln, Z¹⁴        is Asp or Gly, Z¹⁵ is Arg or Lys, Z¹⁶ is Asp or Gly and Z¹² is        Arg,        or includes a subsequence of formula (III), such as a        subsequence having at least 5 amino acid residues (such as at        least 6, at least 7, at least 8, at least 9, at least 10, at        least 11, at least 12, at least 13, at least 14, at least 15,        and at least 16 amino acid residues). Further, this embodiment        of the second peptide may contain amino acid substitutions which        result in a sequence identity of at least 80% with a        corresponding amino acid sequence found in gp41.

Particularly interesting peptides derived from C2 and gp41, and gp120are set forth in the preamble to the Examples and constitute embodimentsof a second peptide of the peptide multimers to be used according to theinvention.

In certain embodiments of the peptide multimer, the first peptide andthe at least one second peptide are associated via a linker; the linkercan be any peptide linker, such as a glycine, a lysine or an argininelinker, a polyhistidinyl tag, Protein G, and Protein A but it is alsopossible to use a bis-maleimide linker, a disulfide linker, or apolyethylene glycol (PEG) linker. In practice, any linker found usefulin peptide chemistry is also useful as a linker according to the presentinvention. Thus, the invention contemplates the use of “simple” linearpeptides which are conjugated or fused to each other, but also peptidemultimers where the individual peptides derived from C5 and otherregions of gp120 or gp41 are linked via non-peptide linkers e.g.complementary nucleic acids, nucleic acid derivatives or analogues e.g.PNA, LNA. Use of multiple linker types are also within the scope of thepresent invention, and it is e.g. also a part to be used according tothe invention to utilise linear peptides which include intrachaindisulphide linkers.

Particularly interesting peptide multimers to be used according to theinvention are set forth in the preamble to the examples.

In certain embodiments, at least one of the first and at least onesecond peptides in the peptide multimer comprises an N- or C-terminalmodification, such as an amidation, acylation, or acetylation.

Since the peptide multimers are contemplated as vaccine agents ordiagnostic agents, they are in certain embodiments coupled to a carriermolecule, such as an immunogenic carrier. The peptides of the peptidemultimers may thus be linked to other molecules either as recombinantfusions (e.g. via CLIP technology) or through chemical linkages in anoriented (e.g. using heterobifunctional cross-linkers) or nonorientedfashion. Linking to carrier molecules such as for example diphtheriatoxin, latex beads (convenient in diagnostic and prognosticembodiments), and magnetic beads (also convenient in diagnostic andprognostic embodiments), polylysine constructs etc, are all possiblecarrier molecules to be used according to the invention.

The immunogenic carrier is conveniently selected from carrier proteinssuch as those conventionally used in the art (e.g. diphtheria or tetanustoxoid, KLH etc.), but it is also possible to use shorter peptides(T-helper epitopes) which can induce T-cell immunity in largerproportions of a population. Details about such T-helper epitopes cane.g. be found in WO 00/20027, which is hereby incorporated by referenceherein—all immunolgic carriers and “promiscuous” (i.e. universal)T-helper epitopes discussed therein are useful as immunogenic carriersin the present invention.

In certain embodiments, the carrier is a virus like particle, i.e. aparticle sharing properties with virions without being infectious. Suchvirus-like particles may be provided chemically (e.g. Jennings andBachmann Ann Rev Pharmacol. Toxicol. 2009. 49:303-26 Immunodrugs:Therapeutic VLP-based vaccines for chronic diseases) or using cloningtechniques to generate fusion proteins (e.g. Peabody et al. J. Mol.Biol. 2008; 380: 252-63. Immunogenic display of diverse peptides onvirus-like particles of RNA phage MS2). Another example is “Remune”, anHIV vaccine originally made by Immune Response Corporation, whichconsists of formalin inactivated HIV that has been irradiated to destroythe viral genome. The company was started by Jonas Salk who used thesame technique to generate the killed polio vaccine in widespread usetoday. However, on fixation of HIV, gp120 fell off leaving only gp41 onthe virion surface. This opens for the possibility of directly admixingC5-derived peptides disclosed herein with Remune particles, because itshould still be possible to obtain the binding between C5 and gp41 on aRemune particle.

Embodiments of the aspect related to peptide multimers also includethose wherein the first peptide is selected from the group consisting ofSEQ ID NO:1, 2, 3, 4, and 5 or a fragment thereof, or the inverso-,retro- or retro-inverso form of a peptides selected from SEQ ID NO:1, 2,3, 4, and 5 or a fragment thereof, and wherein the second peptide isselected from the group consisting of SEQ ID NO:6, 7, 8, 9, 10, 11, 12,13, or 46 or a fragment thereof or the inverso-, retro- or retro-inversoform of a peptides selected from SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13,or 46 or a fragment thereof. As mentioned above, in such a case thefragment may be very short, as long as the peptide multimer provides forthe ability to induce antibodies which will stabilise associationbetween C5 and gp41 and/or C2. A number of interesting peptide multimersof the present invention are listed in the Preamble to the Examples.

In an embodiment, the peptide multimer to be used according to theinvention comprises at most 70 amino acids, such as the most 69, at most68, at most 67, at most 66, at most 65, at most 64, at most 63, at most62, at most 61, at most 60, at most 59, at most 58, at most 57, at most56, at most 55, at most 54, at most 53, at most 52, at most 51, at most50, at most 49, at most 48, at most 47, at most 46, at most 45, at most44, at most 43, at most 42, at most 41, at most 40, at most 39, at most38, at most 37, at most 36, at most 35, at most 34, at most 33, at most32, at most 31, at most 30, at most 29, at most 28, at most 27, at most26, at most 25, at most 24, at most 23, at most 22, at most 21, at most20, at most 19, at most 18, at most 17, at most 16, at most 15, at most14, at most 13, at most 12, at most 11, at most 10, at most 9, at most8, and at most 7 amino acids.

In an embodiment, the peptide multimer to be used according to theinvention comprises at least 6 amino acid residues, such as at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29, atleast 30, at least 31, at least 32, at least 33, at least 34, at least35, at least 36, at least 37, at least 38, at least 39, at least 40, atleast 41, at least 42, at least 43, at least 44, at least 45, at least46, at least 47, at least 48, at least 49, at least 50, at least 51, atleast 52, at least 53, at least 54, at least 55, at least 56, at least57, at least 58, at least 59, at least 60, at least 61, at least 62, atleast 63, at least 64, at least 65, at least 66, at least 67, at least68, and at least 69 amino acid residues.

In one embodiment, the peptide multimer to be used according to theinvention consists of 6 amino acid residues or 7 amino acid residues or8 amino acid residues or 9 amino acid residues or 10 amino acid residuesor 11 amino acid residues or 12 amino acid residues or 13 amino acidresidues or 14 amino acid residues or 15 amino acid residues or 16 aminoacid residues or 17 amino acid residues or 18 amino acid residues or 19amino acid residues or 20 amino acid residues or 21 amino acid residuesor 22 amino acid residues or 23 amino acid residues or 24 amino acidresidues or 25 amino acid residues or 26 amino acid residues or 27 aminoacid residues or 28 amino acid residues or 29 amino acid residues or 30amino acid residues or 31 amino acid residues or 32 amino acid residuesor 33 amino acid residues or 34 amino acid residues or 35 amino acidresidues or 36 amino acid residues or 37 amino acid residues or 38 aminoacid residues or 39 amino acid residues or 40 amino acid residues or 41amino acid residues or 42 amino acid residues or 43 amino acid residuesor 44 amino acid residues or 45 amino acid residues or 46 amino acidresidues or 47 amino acid residues or 48 amino acid residues or 49 aminoacid residues or 50 amino acid residues or 51 amino acid residues or 52amino acid residues or 53 amino acid residues or 54 amino acid residuesor 55 amino acid residues or 56 amino acid residues or 57 amino acidresidues or 58 amino acid residues or 59 amino acid residues or 60 aminoacid residues or 61 amino acid residues or 62 amino acid residues or 63amino acid residues or 64 amino acid residues or 65 amino acid residuesor 66 amino acid residues or 67 amino acid residues or 68 amino acidresidues or 69 amino acid residues or 70 amino acid residues.

In one embodiment, the peptide multimer to be used according to theinvention is selected from the group consisting of disulphide linkedpeptides between SEQ ID NO:28 and any one of SEQ ID NOs: 29, 31, and 33,between SEQ ID NO:30, and any one of SEQ ID NO:29, 31, and 33, orbetween SEQ ID NO:32 and any one of SEQ ID NO:29, 31, and 33; orselected from the group consisting of cysteine-lysine linked peptidesbetween SEQ ID NO:38 and any one of SEQ ID NO:39, SEQ ID NO:40; SEQ IDNO:42, SEQ ID NO:43, and SEQ ID NO:68, or between SEQ ID NO:41 and anyone of SEQ ID NO:39, SEQ ID NO:40; SEQ ID NO:42, and SEQ ID NO:43.

In one embodiment, the peptide multimer to be used according to theinvention is selected from the group consisting of:

CGGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 28) |CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR, (SEQ ID NO: 29)CGGAKRRVVGGAKRRVVGGQREKR (SEQ ID NO: 30) | CGGGDQQLLGGAEEEIVGGIEEEGG,(SEQ ID NO: 31)  CGGAEEEVVGGDQQLL (SEQ ID NO: 32)  | GCGGAKRRVVGGAKRRVV,(SEQ ID NO: 33) GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)         |         GKGGIEEEGGRDRDRGGEQDRDR, (SEQ ID NO: 39)GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)          |        GKGGIEEEGGERDRDRGGQDRDR, (SEQ ID NO: 40)GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO: 41)          |        GKGGIEEEGGQDRDRGGRDRDR, (SEQ ID NO: 42)GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO: 41)             |        GKGGIEEEGGEQDRDRGGERDRD (SEQ ID NO: 43) andGAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)             |        GKGGIEEEGGRDRDRGGQDRDR (SEQ ID NO: 68)

In one embodiment, the peptide multimer to be used according to theinvention is(H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys(2-oxo-ethyl)-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH2)(H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH2),acetate salt (amide bond between Cys(2-oxo-ethyl)10 (A-chain) and Lys2(B-chain)).

Another aspect relates to the use of an immunogenic composition (such asa vaccine composition) comprising a composition described herein incombination with a pharmaceutically acceptable diluent or vehicle andoptionally one or more immunological adjuvant.

In some aspects the present invention relates to the use of one or morepeptide that stimulate a cell-mediated immune response, such as with atleast one HIV-specific peptide selected from the group of amino acidsequences:

(SEQ ID NO: 47) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Ala Xaa₈ Xaa₉ Gln Thr ProTrp Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Val Xaa₂₀;Wherein Xaa in position 1 of the peptide derivate is Lys or Arg,Xaa in position 2 is Ala, Gly, Ser or Arg,Xaa in position 3 is Leu or Met,Xaa in position 4 is Gly or Arg,Xaa in position 5 is Pro, Thr, Val, Ser, Gln or Ala,Xaa in position 6 is Gly, Ala, Lys, Arg, Gln or Glu,Xaa in position 8 is Thr or Ser,Xaa in position 9 is Leu or Ile,Xaa in position 14 is Thr, Ser or Val,Xaa in position 15 is Ala or Ser,Xaa in position 16 is Cys or Ser,Xaa in position 17 is Gln or LeuXaa in position 18 is Gly, Glu or Arg, andXaa in position 20 is Gly or Arg;

(SEQ ID NO: 50) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Gly Leu Asn Pro Leu Val[Gly]_(n) Xaa₁₂ Xaa₁₃ Tyr Xaa₁₅ Pro Xaa₁₇ Xaa₁₈ Ile Leu Xaa₂₁ Xaa₂₂wherein Xaa in position 1 is Arg, Lys, Asp or noneXaa in position 2 is Trp, Gly, Lys or Arg,Xaa in position 3 is Ile, Leu, Val or MetXaa in position 4 is Ile, Val or LeuXaa in position 5 Leu, Met, Val or ProXaa in position 12 is Arg, LysXaa in position 13 is Met or Leu,Xaa in position 15 is Ser, Cys or Gln,Xaa in position 17 is Thr, Val, Ile, Ser or Ala,Xaa in position 18 is Ser, Gly or Thr,Xaa in position 21 is Asp, Glu, Cys or Gly,Xaa in position 22 is Gly or none, andn=0, 1, 2 or 3;

(SEQ ID NO: 55) Xaa₁ Xaa₂ Xaa₃ Pro Ile Pro Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁Xaa₁₂ [Gly]_(n) Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁Xaa₂₂ Xaa₂₃ Xaa₂₄wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or noneXaa in position 2 is Asn, Ala or LysXaa in position 3 is Pro, Gln, Gly, Ile or LeuXaa in position 7 is Val or AlaXaa in position 8 is Gly or LysXaa in position 9 is Glu, Asp, Lys, Phe or ThrXaa in position 10 is Ile, Met, Val or LeuXaa in position 11 is Tyr, Leu or noneXaa in position 12 is Ser or noneXaa in position 13 is Arg or noneXaa in position 14 is Asp, Arg, Trp, Ala or noneXaa in position 15 is Ile or noneXaa in position 16 is Tyr or noneXaa in position 17 is Lys or ArgXaa in position 18 is Arg, Lys or AspXaa in position 19 is Trp or GlyXaa in position 20 is Ile, Met, Val, Gln or AlaXaa in position 21 is Ile, Val or AlaXaa in position 22 is Leu, Met or ValXaa in position 23 is Gly or CysXaa in position 24 is Leu or none,n=1, 2 or 3, and

(SEQ ID NO: 61) Xaa₁ Xaa₂ Ile Ile Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Leu Xaa₁₁[Gly]_(n) [Arg]_(m) Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅wherein the Xaa in position 1 is Pro, Lys, Arg or noneXaa in position 2 is Glu, Arg, Phe or LysXaa in position 5 is Pro or ThrXaa in position 6 is Met, Thr or NleuXaa in position 7 is Phe or LeuXaa in position 8 is Ser, Thr, Ala or MetXaa in position 9 is Ala, Glu or LeuXaa in position 11 is Ser or noneXaa in position 12 is Ala, Arg or noneXaa in position 13 is Ile, Leu or noneXaa in position 14 is Ser, Ala, Leu or noneXaa in position 15 is Tyr, Glu or AspXaa in position 16 is Gly or AspXaa in position 17 is Ala or LeuXaa in position 18 is Thr, Ile, Val, Leu or Asn,Xaa in position 19 is Pro, Thr or SerXaa in position 20 is Tyr, Phe, Nleu, His or GlnXaa in position 21 is Asp, Asn, Leu or AlaXaa in position 22 is Leu, Ile, Val or AsnXaa in position 23 is Asn, Tyr, Cys or GlyXaa in position 24 is Thr, Met, Ile, Ala, Val or noneXaa in position 25 is Gly or nonen=1, 2 or 3 and m=0, 1, 2 or 3 independent of each other,the terminal ends of each HIV specific peptide may be free carboxyl- oramino groups, amides, acyls, acetyls or salts thereof.

In some embodiments two or more of the Cys residues of said HIV-specificpeptide may form part of an intrachain- or interchain disulphidebinding, a —S—(CH₂)_(p)—S— or a —(CH₂)_(p)— bridge wherein p=1-8optionally intervened by one or more heteroatoms such as O, N and Sand/or the said peptide sequences are immobilized to a solid support.

In some embodiments the amino acid sequence of SEQ ID NO:47 is selectedfrom the groups of SEQ ID NO:48 and SEQ ID NO:49.

In some embodiments the amino acid sequence of SEQ ID NO:50 is selectedfrom the groups of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ IDNO:54.

In some embodiments the amino acid sequence of SEQ ID NO:55 is selectedfrom the groups of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59 and SEQ ID NO:60.

In some embodiments the amino acid sequence of SEQ ID NO:61 is selectedfrom the groups of SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ IDNO:65 and SEQ ID NO: 66.

In some embodiments the at least one HIV-specific peptide comprises atleast, two, three, or four peptides selected from each of the groups ofSEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:55 and SEQ ID NO:61.

In some embodiments the at least one HIV-specific peptide consist of orcomprises the peptides of the SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:57and SEQ ID NO:64. In some embodiments the one or more peptide is in theform of an acetate salt.

In some embodiments one, two, three or four peptides are used in thetherapeutic HIV-1 immunization phase.

In some embodiments the all four peptide as acetate salts are used inthe therapeutic HIV-1 immunization phase.

In some embodiments the peptides have amide C-terminal ends of formula—C(O)NH2, or acetate salts thereof.

In some embodiments all four peptide are used in the ratio of 1:1:1:1w/w.

In some embodiments one, two, three or four peptide acetate salts are ina dissolved liquid state. In some embodiments this liquid is water.

Specific Aspects and Embodiments of the Invention

One aspect of the present invention relates to the use of one or moreHIV-specific peptide as defined above.

In certain embodiments, peptides comprise an N- or C-terminalmodification, such as an amidation, acylation, or acetylation. When theC-terminal end of a peptide is an amide, suitable amides included thosehaving the formula —C(O)—NR^(x)R^(y), wherein R^(x) and R^(y) areindependently selected from hydrogen and C₁₋₆ alkyl, which alkyl groupmay be substituted with one of more fluoro atoms, for example —CH₃,—CH₂CH₃ and —CF₃, a particular amide group which may be mentioned is—C(O)NH₂. When the N-terminal end of the peptide is acetylated, suitableacetylated N-terminal ends include those of formula —NH—C(O)R^(z),wherein R^(z) is hydrogen, C₁₋₆ alkyl, which alkyl group may besubstituted with one of more fluoro atoms, for example —CH₃, —CH₂CH₃ and—CF₃, or phenyl.

Since the peptides are contemplated as vaccine agents or diagnosticagents, they are in certain embodiments coupled to a carrier molecule,such as an immunogenic carrier. The peptides may thus be linked to othermolecules either as recombinant fusions (e.g. via CLIP technology) orthrough chemical linkages in an oriented (e.g. using heterobifunctionalcross-linkers) or nonoriented fashion. Linking to carrier molecules suchas for example diphtheria toxin, latex beads (convenient in diagnosticand prognostic embodiments), and magnetic beads (also convenient indiagnostic and prognostic embodiments), polylysine constructs etc, areall possible according to the invention.

The immunogenic carrier is conveniently selected from carrier proteinssuch as those conventionally used in the art (e.g. diphtheria or tetanustoxoid, KLH etc.), but it is also possible to use shorter peptides(T-helper epitopes) which can induce T-cell immunity in largerproportions of a population. Details about such T-helper epitopes cane.g. be found in WO 00/20027, which is hereby incorporated by referenceherein—all immunolgic carriers and “promiscuous” (i.e. universal)T-helper epitopes discussed therein are useful as immunogenic carriersin the present invention.

In certain embodiments, the carrier is a virus like particle, i.e. aparticle sharing properties with virions without being infectious. Suchvirus-like particles may be provided chemically (e.g. Jennings andBachmann Ann. Rev. Pharmacol. Toxicol. 2009. 49:303-26 Immunodrugs:Therapeutic VLP-based vaccines for chronic diseases) or using cloningtechniques to generate fusion proteins (e.g. Peabody et al. J. Mol.Biol. 2008; 380: 252-63. Immunogenic display of diverse peptides onvirus-like particles of RNA phage MS2). Another example is “Remune”, anHIV vaccine originally made by Immune Response Corporation, whichconsists of formalin inactivated HIV that has been irradiated to destroythe viral genome. The company was started by Jonas Salk who used thesame technique to generate the killed polio vaccine in widespread usetoday.

Preparation of immunogenic compositions includes the use ofstate-of-the-art constituents such as immunological adjuvants. Apartfrom these adjuvants, which are detailed, by way of example, below,immunogenic compositions are prepared as generally taught in the art:

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like, and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines; cf. thedetailed discussion of adjuvants below.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously, intracutaneously, intradermally,subdermally or intramuscularly. Additional formulations which aresuitable for other modes of administration include suppositories and, insome cases, oral, nasal, buccal, sublingual, intraperitoneal,intravaginal, anal, epidural, spinal, and intracranial formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10% (w/w), preferably 1-2% (w/w). Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and may contain 10-95% (w/w) of activeingredient, preferably 25-70% (w/w).

The peptides may be formulated into a vaccine as neutral or salt forms.Pharmaceutically acceptable salts include acid addition salts (formedwith the free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed with the free carboxyl groups may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and organic bases such as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to mount an immune response, and the degree of immunity desired.Suitable dosage ranges are of the order of several hundred micrograms ofactive ingredient per vaccination with a preferred range from about 0.1μg to 2,000 μg (even though higher amounts in the 1-10 mg range arecontemplated), such as in the range from about 0.5 μg to 1,000 μg,preferably in the range from 1 μg to 500 μg and especially in the rangefrom about 10 μg to 100 μg. Suitable regimens for initial administrationand booster shots are also variable but are typified by an initialadministration followed by subsequent inoculations or otheradministrations.

Some of the peptides are sufficiently immunogenic in a vaccine, but forsome of the others the immune response will be enhanced if the vaccinefurther comprises an adjuvant substance. The immunogenic moleculesdescribed herein can therefore be formulated with adjuvants:

The adjuvants to be combined are known to induce humoral responses andinclude: i) Salt suspensions (e.g. varieties of salts containingaluminum ions or calcium ions), ii) Oil-in-water emulsions (e.g.varieties of squalane-based or squalene-based emulsions), iii)Water-in-oil emulsions (e.g. Montanide ISA51 or ISA720), iv) Neutralliposomes, v) Cationic liposomes, vi) Microspheres, vii)Immunostimulating complexes (e.g. ISCOMs or ISCOMATRIX), viii)Pattern-recognition receptor agonists (e.g. agonists for C-type lectinreceptors (CLRs), NOD-like receptors (NLRs), RIG-like helicases (RLHs),Triggering receptor expressed on myeloid cells (TREMs) and Toll-likereceptors (TLRs)), ix) Saponins (i.e. Any saponin derived from Quillajasaponaria or Platycodon grandiflorum), x) Virosomes/Virus-likeparticles, xi) Enterotoxins (i.e. Cholera toxin, CTA1-DD or Esherichiacoli heat-labile enterotoxin), and combinations thereof.

For a further enhancement of the vaccine antigenic properties, theycould be combined with a well known adjuvant with an oral immunemodulant or adjuvant such as a Cox-2 inhibitor or a immunomodulatingcompound.

A further aspect of the invention is the use of the vaccine combinedwith adjuvant, with one or more further therapeutic agents, such as an(oral) immunomodulating agent and/or a second reservoir purging agent.

The terms “therapeutic agent”, such as “immunomodulating agent” or virusreservoir purging agent as used herein, includes but is not limited tocytokines, such as interferons, monoclonal antibodies, such as anti-PD1antibodies, cyclophosphamide, Thalidomide, Levamisole, and Lenalidomide.

“A virus reservoir purging agent”, includes but is not limited toauranofin, IL-7, prostratin, bryostatin, HDAC inhibitors, such asvorinostat, Disulfiram and any suitable agent disclosed in any one ofWO2013050422, WO2012051492 A3 and in Barton et al., ClinicalPharmacology & Therapeutics (2013); 93 1, 46-561, including but notlimited to a NF-kappa-B-inducer selected from the group comprising: PMA,prostratin, bryostatin and TNF-alpha, and/or b) a histone deacetylaseinhibitor selected from the different families (hydroxamates, cyclicpeptides, aliphatic acids, and benzamides) including: TSA, SAHA, MS-275,aminosuberoyl hydroxamic acids, M-Carboxycinnamic acid bishydroxamate,LAQ-824, LBH-589, belinostat (PXD-101), Panobinostat (LBH-589), acinnamic hydroxamic acid analogue of M-carboxycinnamic acidbishydroxamate, IF2357, aryloxyalkanoic acid hydroxamides, depsipeptide,apicidin, cyclic hydroxamic acid-containing peptide group of molecules,FK-228, red FK, cyclic peptide mimic linked by an aliphatic chain to ahydroxamic acid, butyrate, phenylbutyrate, sodium butyrate, valproicacid, pivaloyloxymethyl butyrate, 5 NOX-275, and MGCD0103. Any of theabove virus reservoir purging agents may be used alone or in combinationwith any one other suitable virus reservoir purging agent, such as withanother class of HIV inducers.

DNA methylation, probably together with repressive histonemodifications, may also contribute to a “lock” in a silent state of theprovirus and makes its return to an active state difficult. Theseobservations suggest that HDAC or HMT or DNA methylation inhibitorstogether with efficient cART constitute good anti-latency drugcandidates aimed at reducing/eliminating the pool of latent reservoirsto a level bearable by the host immune system.

Accordingly suitable immunomodulatory compounds or purging agents may beDNA methylation inhibitors selected from the two classes (non-nucleosideand nucleoside demethylating agents) including: 5-azacytidine(azacitidine), Sinefungin, 5-aza-2′-deoxycytidine (5-aza-CdR,decitabine), 1-3-Darabinofuranosyl-5-azacytosine (fazarabine) anddihydro-5-azacytidine (DHAC), 5-fluorodeoxycytidine (FdC),oligodeoxynucleotide duplexes containing 2-H pyrimidinone, zebularine,antisense oligodeoxynucleotides (ODNs), MG98,(−)-epigallocatechin-3-gallate, hydralazine, procaine and procainamide.

Other suitable immunomodulatory compounds or purging agents to be usedaccording to the present invention includes histone deacetylaseinhibitor selected from the different families of HDACI (hydroxamates,cyclic peptides, aliphatic acids, and benzamides) including TSA, SAHA,MS-275, aminosuberoyl hydroxamic acids, M-Carboxycinnamic acidbishydroxamate, LAQ-824, LBH-589, belinostat (PXD-101), Panobinostat(LBH-589), a cinnamic hydroxamic acid analogue of M-carboxycinnamic acidbishydroxamate, IF2357, aryloxyalkanoic acid hydroxamides, depsipeptide,apicidin, cyclic hydroxamic acid-containing peptide group of molecules,FK-228, red FK, cyclic peptide mimic linked by an aliphatic chain to ahydroxamic acid, butyrate, phenylbutyrate, sodium butyrate, valproicacid, pivaloyloxymethyl butyrate, 5 NOX-275, and MGCD0103.

Other suitable immunomodulatory compounds or purging agents to be usedaccording to the present invention includes histone methyltransferaseinhibitors (chaetocin and BIX-01294); Inhibitors of Enhances of Zeste 2(EZH2)—such as 3-deazaneplanocin A (DZNep) used alone or in combinationwith other classes of immunomodulatory compounds or purging agents.

Other suitable adjuvants include response-selective C5a agonists, suchas EP54 and EP67 described in Hung C Y et al. An agonist of humancomplement fragment C5a enhances vaccine immunity against Coccidioidesinfection. Vaccine (2012) and Kollessery G et al. Tumor-specific peptidebased vaccines containing the conformationally biased,response-selective C5a agonists EP54 and EP67 protect against aggressivelarge B cell lymphoma in a syngeneic murine model. Vaccine (2011) 29:5904-10.

Various methods of achieving adjuvant effect for the vaccine are thusknown. General principles and methods are detailed in “The Theory andPractical Application of Adjuvants”, 1995, Duncan E. S. Stewart-Tull(ed.), John Wiley & Sons Ltd, ISBN 0-471-95170-6, and also in “Vaccines:New Generation Immunological Adjuvants”, 1995, Gregoriadis G et al.(eds.), Plenum Press, New York, ISBN 0-306-45283-9, both of which arehereby incorporated by reference herein, but a number of laterpublications also deal with the technology of incorporating adjuvants:Roestenberg M et al., PLoS One. 2008; 3(12):e3960. Epub 2008 Dec. 18;Relyveld E and Chermann J C, Biomed Pharmacother. 1994; 48(2):79-83; HsuF J et al., Blood. 1997 May 1; 89(9):3129-35; Galli G et al., Proc NatlAcad Sci USA. 2009 May 12; 106(19):7962-7. Epub 2009 Apr. 27; Bojang K Aet al., Lancet. 2001 Dec. 8; 358(9297):1927-34; Odunsi K et al., ProcNatl Acad Sci USA. 2007 Jul. 31; 104(31):12837-42. Epub 2007 Jul. 25;Patel G B and Sprott G D; Crit Rev Biotechnol. 1999; 19(4):317-57.Review; Agger E M et al., PLoS One. 2008 Sep. 8; 3(9):e3116; Kirby D Jet al. J Drug Target. 2008 May; 16(4):282-93; Florindo H F et al.,Vaccine. 2008 Aug. 5; 26(33):4168-77. Epub 2008 Jun. 17; Sun H X et al.,Vaccine. 2009 May 28; Guy B, Nat Rev Microbiol. 2007 July; 5(7):505-17.Review.; Vandepapeliére P et al., Vaccine. 2008 Mar. 4; 26(10):1375-86.Epub 2008 Jan. 14; Ghochikyan A et al. Vaccine. 2006 Mar. 20;24(13):2275-82. Epub 2005 Dec. 5; Xie Y et al., Vaccine. 2008 Jun. 25;26(27-28):3452-60. Epub 2008 May 1; Chung Y C et al., Vaccine. 2008 Mar.28; 26(15):1855-62. Epub 2008 Feb. 25; Maier M et al., Vaccine. 2005Oct. 25; 23(44):5149-59; Sundling C et al., J Gen Virol. 2008 Dec.;89(Pt 12):2954-64.

In the methods of the invention the one or more peptide to stimulate acell-mediated immune response and/or a compound that stimulate a humoralresponse in a human, may be administered in combination with one or morefurther immunomodulatory compound and/or a reservoir purging agent, suchas a histone deacetylase (HDAC) inhibitor.

The term “immunomodulating compound” or “reservoir purging agent” asused herein, includes but is not limited to cytokines, such asinterferons, monoclonal antibodies, such as ant-PD1 antibodies,cyclophosphamide, Thalidomide, Levamisole, and Lenalidomide.

“A reservoir purging agent”, includes but is not limited to auranofin,IL-7, prostratin, bryostatin, HDAC inhibitors, such as vorinostat, andDisulfiram, and the further agents described herein.

The failure of antiretroviral therapy (ART) to eradicate HIV-1 infectionlies in the observation that HIV-1 remains quiescent in latentreservoirs. Latently infected resting CD4+ cells (either naive or longlived memory cells) carry transcriptionally silent HIV-1 and representthe predominant reservoir of HIV-1 infection. Other cells may also actas reservoirs (Reviewed in Alexaki et al., 2008, Curr. HIV Res.6:388-400), such as macrophages, dendritic cells and astrocytes (whereHIV-1 infection occurs via a CD4-independent mechanism). It is theselatent reservoirs that represent the major challenge to eradication ofHIV-1 infection. Approaches towards eradication include attempts topurge reservoirs by selective activation of latently infected cells(such as memory cells) in the presence of ART such that released virusmay not infect and replicate in neighbouring cells (Richman et al.,2009, Science 323:1304-1307). Agents include histone deacetylaseinhibitors, cytokines, such as IL-2 and IL-7, as well as bryostatin, theprotein kinase C activator (Kovochich et al., 2011, PLoS ONE 6(4):e18270). Therapeutic vaccines have the advantage of being able topenetrate sanctuary sites less well accessed by ART such as lymphoidtissue (Pantaleo et al., 1991, Proc. Natl. Acad. Sci. USA 88:9838-42;Fox et al., 1991, J. Infect. Dis. 164:1051-57) and the central nervoussystem (Alexaki et al., 2008, Curr. HIV Res. 6:388-400), that representregions for viral persistence. This relates to therapeutic interventionstargeting both the virus itself as well as HIV-associated immuneactivation.

A number of studies have been conducted with the aim of providingcompounds that can safely and effectively be used to treat diseasesassociated with abnormal production of TNF-α. See, e.g., Marriott, J.B., et al, Expert Opin. Biol. Ther. (4): 1-8 (2001); G. W. Muller, etal, Journal of Medicinal Chemistry, 39(17): 3238-3240 (1996); and G. W.Muller, et al, Bioorganic & Medicinal Chemistry Letters, 8: 2669-2674(1998). Some studies have focused on a group of compounds selected fortheir capacity to potently inhibit TNF-α production by LPS stimulatedPBMC. L. G. Corral, et al, Ann. Rheum. Dis., 58 (suppl I): 1107-1113(1999). These compounds, often referred to as immunomodulatorycompounds, show not only potent inhibition of TNF-α, but also markedinhibition of LPS induced monocyte IL1B and IL12 production. LPS inducedIL6 is also inhibited by immunomodulatory compounds, albeit partially.These compounds are potent stimulators of LPS induced IL10. Particularexamples include, but are not limited to, the substituted2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoles as described in U.S. Pat.No. 6,281,230 and U.S. Pat. No. 6,316,471. Monocyte/macrophage functionis part of the Innate Immune System that serves as a first line ofdefense against an infection. By modulating the host's monocytes andmacrophages, immunomodulatory compounds can change the dynamics of theresponse to a viral infection, such as influenza.

Histone deacetylases (HDAC) are a class of enzymes that remove acetylgroups from N-acetylated lysines amino acid on histone proteins.Currently 18 HDACs have been identified in mammals. They have beendivided into four classes based on cellular localization, function, andsequence similarity. Class I includes HDACs 1, 2, 3, and 8 which arefound primarily in the nucleus. Class II HDACs (HDACs 4, 5, 6, 7 9, and10) are found primarily in the cytoplasm but may be able to shuttlebetween the nucleus and the cytoplasm; class IIa comprises four HDACs(HDACs 4, 5, 7 and 9) while class IIb comprises two HDACs (HDACs 6 and10) which are expressed only in the cytoplasm. HDAC11, which isubiquitously expressed, shares sequence similarities with both class Iand class II HDACs and represents Class IV. Class III (also called“sirtuin family”) groups NAD+-dependent proteins which do not actprimarily on histones.

The immunomodulatory compounds may be selected from anti-PD1 antibodies,such as MDX-1106 (Merck), THALOMID® (thalidomide), anti-PD1 antibodies,cyclophosphamide, Levamisole, lenalidomide, CC-4047 (pomalidomide),CC-11006 (Celgene), and CC-10015 (Celgene), and immunomodulatorycompound described in any one of WO2007028047, WO2002059106, andWO2002094180. The immunomodulatory compound may be selected from4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. Inparticular the immunomodulatory compound is lenalidomide. Theimmunomodulatory compound may be enantiomerically pure. The secondreservoir purging agent, such as a histone deacetylase (HDAC) inhibitor,may be selected from M344(4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide), chidamide(CS055/HBI-800), 4SC-202, (4SC), Resminostat (4SC), hydroxamic acidssuch as vorinostat (SAHA), belinostat (PXD101), LAQ824, trichostatin Aand panobinostat (LBH589); benzamides such as entinostat (MS-275),CI994, and mocetinostat (MGCD0103), cyclic tetrapeptides (such astrapoxin, such as trapoxin B), and the depsipeptides, such as romidepsin(Istodax® (Celgene)), electrophilic ketones, and the aliphatic acidcompounds such as phenylbutyrate, valproic acid, Oxamflatin, ITF2357(generic givinostat), Apicidin, MC1293, CG05, and CG06; compounds thatactivate transcription factors including NF-KappaB, Prostratin,auranofin, bryostatin, a nontumorigenic phorbol ester, DPP(12-deoxyphorbol-13-phenylacetate), PMA, and Phorbol 12-myristate13-acetate (PMA); Compounds that activate HIV mRNA elongation includingP-TEF-b kinase and hexamethylbisacetamide (HMBA); IL-7; T-cellstimulating factors including anti-CD3/CD28− T-cell stimulating Ab's;Kinase inhibitors including Tyrphostin A, Tyrphostin B, and TyrphostinC; PTEN (phosphatase and tensin homologue) gene inhibitors includingSF1670 (Echelon Bioscience), Disulfiram (DSF), an inhibitor ofacetaldehyde dehydrogenase, Protein Tyrosine Phosphatase Inhibitorsincluding bpV(HOpic), bpV(phen), and bpV(pic) (Calbiochem; EMDMillipore), Toll-like receptors agonists including Toll-like receptor-9(TLR9) and Toll-like receptor-7 (TLR9) agonists, quercetin, lipoic acid,sodium butyrate, TNF-alpha, PHA, Tat.

In the methods of the invention the components of the at least oneHIV-specific peptide and/or the one or more further therapeuticallyactive agents, may be administered simultaneously, sequentially orseparately in any order.

Thus the invention provides a pharmaceutical composition comprising one,two or more components of the at least one HIV-specific peptide and/orthe one or more further therapeutically active agents optionally incombination with one or more pharmaceutically acceptable adjuvants,diluents or carriers.

Similarly, the invention also provides a combination product comprisingof components of the at least one HIV-specific peptide and/or the one ormore further therapeutically active agents, wherein each of component isformulated in admixture with a pharmaceutically-acceptable adjuvant,diluent or carrier. In this aspect of the invention, the combinationproduct may be either a single (combination) pharmaceutical formulationor a kit-of-parts. In a kit-of-parts some or all of the components maybe formulated separately and may each be provided in a form that issuitable for administration in conjunction with the other(s).

The component(s) may also be provided for use, e.g. with instructionsfor use, in combination with one or more further component(s) as definedabove.

The peptides for use in the invention may be produced syntheticallyusing art recognised methods. Further details for the syntheticproduction of such peptides are found in the Examples. Alternatively thepeptides may be produced recombinantly. When recombinantly producing thepeptides for use in the invention by means of transformed cells, it isconvenient, although far from essential, that the expression product iseither exported out into the culture medium or carried on the surface ofthe transformed cell.

When an effective producer cell has been identified it is preferred, onthe basis thereof, to establish a stable cell line which carries thevector of the invention and which expresses the nucleic acid fragment ofthe invention. Preferably, this stable cell line secretes or carries thepeptide expression product, thereby facilitating purification thereof.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with the hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., 1977). The pBR322 plasmid containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR plasmid, or othermicrobial plasmid or phage must also contain, or be modified to contain,promoters which can be used by the prokaryotic microorganism forexpression.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase) and lactose promoter systems(Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and atryptophan (trp) promoter system (Goeddel et al., 1979; EP-A-0 036 776).While these are the most commonly used, other microbial promoters havebeen discovered and utilized, and details concerning their nucleotidesequences have been published.

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used, and also here the promoter should be capable ofdriving expression. Saccharomyces cerevisiase, or common baker's yeastis the most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example, is commonly used(Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al.,1980).

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also incorporated into the expression vector 3′ of thesequence desired to be expressed to provide polyadenylation of the mRNAand termination.

Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions are the promoter region for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedglyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Any plasmid vector containing ayeast-compatible promoter, origin of replication and terminationsequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. Examples of such useful host cell lines are VERO and HeLacells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293,Spodoptera frugiperda (SF) cells, Drosophila melanogaster cell lines(such as Schneider 2 (S₂)), and MDCK cell lines.

Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment which also contains the SV40 viral origin of replication (Fierset al., 1978). Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BglI site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired gene sequence, provided such control sequences are compatiblewith the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., other Polyoma viruses, Adeno, VSV, BPV) or may beprovided by the host cell chromosomal re-application mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

As for routes of administration and administration schemes ofpolypeptide based vaccines which have been detailed above, these arealso applicable for the nucleic acid vaccines of the invention and alldiscussions above pertaining to routes of administration andadministration schemes for polypeptides apply mutatis mutandis tonucleic acids. To this should be added that nucleic acid vaccines canalso be administered intravenously and intraarterially. Furthermore, itis well-known in the art that nucleic acid vaccines can be administeredby use of a so-called gene gun and/or by use of electroporation, andhence also these and equivalent modes of administration are regarded aspart of the present invention.

Under normal circumstances, the nucleic acid fragment is introduced inthe form of a vector wherein expression is under control of a viralpromoter. For more detailed discussions of vectors according to theinvention, cf. the discussion above. Also, detailed disclosures relatingto the formulation and use of nucleic acid vaccines are available, cf.Donnelly J J et al, 1997, Annu. Rev. Immunol. 15: 617-648 and Donnelly JJ et al., 1997, Life Sciences 60: 163-172. Both of these references areincorporated by reference herein.

An alternative of using peptide immunogens or nucleic acid immunogens isthe use of live immunogen technology. This entails administering anon-pathogenic microorganism which has been transformed with a nucleicacid fragment or a vector of the present invention. The non-pathogenicmicroorganism can be any suitable attenuated bacterial strain(attenuated by means of passaging or by means of removal of pathogenicexpression products by recombinant DNA technology), e.g. Mycobacteriumbovis BCG., non-pathogenic Streptococcus spp., E. coli, Salmonella spp.,Vibrio cholerae, Shigella, etc. Reviews dealing with preparation ofstate-of-the-art live vaccines can e.g. be found in Saliou P, 1995, Rev.Prat. 45: 1492-1496 and Walker P D, 1992, Vaccine 10: 977-990, bothincorporated by reference herein. For details about the nucleic acidfragments and vectors used in such live vaccines, cf. the discussionbelow.

As an alternative to bacterial live immunogens, the nucleic acidfragment of the invention can be incorporated in a non-virulent viralvaccine vector such as a vaccinia strain or any other suitable poxvirus.

Normally, the non-pathogenic microorganism or virus is administered onlyonce to a subject, but in certain cases it may be necessary toadminister the microorganism/virus more than once in a lifetime in orderto maintain protective immunity. It is even contemplated thatimmunization schemes as those detailed above for polypeptide vaccinationwill be useful when using live or virus vaccines.

Alternatively, live or virus immunization is combined with previous orsubsequent polypeptide and/or nucleic acid immunization. For instance,it is possible to effect primary immunization with a live or virusvaccine followed by subsequent booster immunizations using thepolypeptide or nucleic acid approach.

PREAMBLE TO EXAMPLES Overview Sequences and Abbreviations C5-Sequences:

(SEQ ID NO: 1) APTKAKRRVVQREKRAV (SEQ ID NO: 2) APTKAKRRVVEREKRAV (SEQID NO: 3) APTRAKRRVVQREKRAV (SEQ ID NO: 4) APTRAKRRVVEREKRAV (SEQ ID NO:5) APTEAKRRVVEREKRAV (SEQ ID NO: 44) WWGCAKRRVCGGAKRRVVQREKRA(underlined amino acid residues in SEQ ID NO: 44 are linked via adisulphide linker; the N-terminal W is preferably a D-amino acid and theC-terminal A may be amidated; the peptide is termed BI450-AdjBT_1, whenhaving these two modifications).

C5-Complex Forming Sequences:

DRPEGIEEEGGERDR (where amino acid 4 can be G and/or where amino acid 5can be R and/or where amino acid 13 can be Q and/or where amino acid 14can be G and/or where amino acid 15 can be K; SEQ ID NO: 6);DRPEGIENNGGERDR (SEQ ID NO: 7 where amino acid 4 can be G and/or whereamino acid 5 can be R and/or where amino acid 13 can be Q and/or whereamino acid 14 can be G and/or where amino acid 15 can be K);DRPEGIENNGGERDRDR (where amino acid 4 can be G and/or where amino acid 5can be R and/or where amino acid 13 can be Q and/or where amino acid 14can be G and/or where amino acid 15 can be K and/or where amino acid 16can be G); SEQ ID NO: 46).

(SEQ ID NO: 8) VERYLKDQQLLG; (SEQ ID NO: 9) VERYLKDEELLG; (SEQ ID NO:10) VERYLKDNNLLG; (SEQ ID NO: 11) QLLLNGSLAEEEIVI (SEQ ID NO: 12)QLLLNGSLAEEEVVIV (SEQ ID NO: 13) QLLLNSLAEEEVVI (SEQ ID NO: 37, alsotermed 204d herein) GGAIVNGSLADDDIVI (SEQ ID NO: 45WWGCIEEEGCGGIEEEGGERDR:underlined amino acid residues are linked via a disulphide linker; theN-terminal W is preferably a D-amino acid and the C-terminal R may beamidated; the peptide is termed BI450-AdjBT_2, when having these twomodifications).

Polypeptides I:

Polypeptides I: (Z-SEQ_(c5)-Z-SEQ_(c5))_(n) n = 1, 2, 3, 4 PolypeptidesII: (Z-SEQ_(cx)-Z-SEQ_(cx))_(n) n = 1, 2, 3, 4

Peptide Complexes:

(Z-SEQ_(c5)-Z-SEQ_(c5))_(n)  |  Bis-maleimide linker  |(Z-SEQ_(cx)-Z-SEQ_(cx))_(n) n = 1, 2, 3, 4 (Z-SEQ_(c5)-Z-SEQ_(c5))_(n) | (Z-SEQ_(cx)-Z-SEQ_(cx))_(n) n = 1, 2, 3, 4

Examples of Polypeptides I can be, but are not Restricted to, theFollowing Sequences:

(SEQ ID NO: 14) APTKAKRGGGAPTRAKRGGGAPTEAKR (SEQ ID NO: 15)RVVEREKGGGAKRRVVGGGRVVQREK (SEQ ID NO: 16) GGAKRRVVGGAKRRVVGQREKRAV (SEQID NO: 17) CGGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 18)GGAKRRVVGGAKRRVVGGQREKR (SEQ ID NO: 19) CGGAKRRVVGGAKRRVVGGQREKR (SEQ IDNO: 20) GGAKRRVVGGAKRRVV (SEQ ID NO: 21) GCGAKRRVVGGAKRRVV

Examples of Polypeptides II can be, but are not Restricted to, theFollowing Sequences:

(SEQ ID NO: 22) GGGDQQLLGGAEEEIVGGIEEEGGERDRDR (SEQ ID NO: 23)CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR (SEQ ID NO: 24) GGDQQLLGGAEEEIVGGGERDR(SEQ ID NO: 25) CGGGDQQLLGGAEEEIVGGIEEEGG (SEQ ID NO: 26)GGAEEEVVGGDQQLL (SEQ ID NO: 27) CGGAEEEVVGGDQQLL

Examples of Disulfide Linked Constructs can be, but are not Restrictedto, the Following Linked Peptide Sequences:

CGGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 28) |CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR (SEQ ID NO: 29) CGGAKRRVVGGAKRRVVGGQREKR(SEQ ID NO: 30) | CGGGDQQLLGGAEEEIVGGIEEEGG (SEQ ID NO: 31) CGGAEEEVVGGDQQLL (SEQ ID NO: 32)  | GCGGAKRRVVGGAKRRVV (SEQ ID NO: 33)

The above disulfide linked constructs may e.g. be synthesised bytitration of 2-pyridinesulfenyl (SPyr)-protected cysteine-containingpeptides with thiol-unprotected peptides. This has proven to be asuperior procedure to selectively generate disulfide-linked peptideheterodimers preventing the formation of homodimers (Schutz A et al.,Tetrahedron, Volume 56, Issue 24, 9 Jun. 2000, Pages 3889-3891). Similarconstructs where SEQ ID NO: 28 is disulphide linked to SEQ ID NOs 31 or33, or where SEQ ID NO: 30 is disulphide linked to SEQ ID NOs: 29 or 33,or where SEQ ID NO: 32 is disulphide linked to SEQ ID NOs: 29 or 31 arealso within the scope of the present invention.

Examples of other linked constructs can be, but are not restricted to,the following linked peptide sequences, which have all been obtainedfrom Bachem (UK) Ltd:

GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)          |  GKGGIEEEGGRDRDRGGEQDRDR (SEQ ID NO: 39)(the peptides are linked via the underlined Cys and Lys residues; theentire construct is termed BI400-B herein).

GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)          |        GKGGIEEEGGERDRDRGGQDRDR (SEQ ID NO: 40)(the peptides are linked via the underlined Cys and Lys residues; theentire construct is termed BI400-Bu1 herein).

GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO: 41)          |        GKGGIEEEGGQDRDRGGRDRDR (SEQ ID NO: 42)(the peptides are linked via the underlined Cys and Lys residues; theentire construct is termed BI400-Bu2 herein).

GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO: 41)          |        GKGGIEEEGGEQDRDRGGERDRD (SEQ ID NO: 43)(the peptides are linked via the underlined Cys and Lys residues; theentire construct is termed BI400-Bu3 herein).

The Cys-Lys linker is typically established in the form of an amide bondbetween (2-oxo-ethyl) derivatized cysteine in one peptide and lysine inthe other peptide.

Similar constructs where SEQ ID NO: 38 is Cys-Lys linked to SEQ ID NOs42 or 43, or where SEQ ID NO: 41 is Cys-Lys linked to SEQ ID NOs: 39 or40 are also within the scope of the present invention.

Small Molecule Inhibitors:

(SEQ ID NO: 34) DQQLL (SEQ ID NO: 35) AKRRVV (SEQ ID NO: 36) AEEEVV

SEQ ID NOs 34-36 are preferably composed partly or completely of D-aminoacids.

One preferred agent capable of stabilising association of the C5 domainof HIV gp120 with the transmembrane domain of gp41 and/or with theconstant C2 domain of gp120 is a compound of the following structure:

(H-Glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-glycyl-glycyl-L-cysteinyl(2-oxo-ethyl)-glycyl-glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-L-glutaminyl-L-arginyl-L-glutamyl-L-lysyl-L-arginyl-L-alanyl-glycyl-L-glutamyl-L-arginyl-L-glutamyl-L-lysyl-L-arginyl-L-alanyl-NH₂)(H-Glycyl-L-lysyl-glycyl-glycyl-L-isoleucyl-L-glutamyl-L-glutamyl-L-glutamyl-glycyl-glycyl-L-arginyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-glycyl-glycyl-L-glutaminyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-NH₂), acetate salt (amide bond between Cys(2-oxo-ethyl)¹⁰ (A-chain) andLys² (B-chain))

This compound may also be referred to as Vacc-C5 with the followingformula:

(H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys(2-oxo-ethyl)-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH₂)(H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH₂),acetate salt (amide bond between Cys(2-oxo-ethyl)¹⁰ (A-chain) and Lys²(B-chain))

This preferred C5 compound consists of two linear peptide amide chainswith 31 amino acids (A-chain) and 22 amino acids (B-chain). Each chainhas a free amino group at the N-terminus and an amide group at theC-terminus. The chains are covalently linked via an amide bond betweenCys(2-oxo-ethyl)¹⁰ of the A-chain and Lys² of the B-chain. All aminoacid residues except the achiral Gly are in the L-configuration.

The preferred C5 compound may be provided as an acetate salt. Thecounter ion acetate is bound in ionic form to basic groups of thepeptide molecule.

HIV-Specific Peptides According to the Invention

The present invention relates to agents capable of stabilising theassociation of the C5 domain of HIV gp120 with the transmembrane domainof gp41 and/or with the constant C2 domain of gp120 in combination withpeptides based on conserved regions of HIV gag p24, antigens in free orcarrier-bound form comprising at least one of the said peptides.

The HIV-specific peptides according to the invention are originatingfrom the four different conserved areas of the HIV-1 core protein p24which are described above, having the properties of maintaining theuniqueness (sensitivity and specificity) of the HIV-1-epitope. Furtherthe new peptides according to the invention possess no recognizedcytotoxic T lymphocyte (CTL) antagonistic effect and shall have at leastone potential CTL epitope.

The HIV-specific peptides, according to the invention, which have metthe above criteria are selected from the following groups;

(SEQ ID NO: 47) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Ala Xaa₈ Xaa₉ Gln Thr ProTrp Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Val Xaa₂₀;Wherein Xaa in position 1 of the peptide derivate is Lys or Arg,Xaa in position 2 is Ala, Gly, Ser or Arg,Xaa in position 3 is Leu or Met,Xaa in position 4 is Gly or Arg,Xaa in position 5 is Pro, Thr, Val, Ser, Gln or Ala,Xaa in position 6 is Gly, Ala, Lys, Arg, Gln or Glu,Xaa in position 8 is Thr or Ser,Xaa in position 9 is Leu or Ile,Xaa in position 14 is Thr, Ser or Val,Xaa in position 15 is Ala or Ser,Xaa in position 16 is Cys or Ser,Xaa in position 17 is Gln or LeuXaa in position 18 is Gly, Glu or Arg, andXaa in position 20 is Gly or Arg;

(SEQ ID NO: 50) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Gly Leu Asn Pro Leu Val[Gly]_(n) Xaa₁₂ Xaa₁₃ Tyr Xaa₁₅ Pro Xaa₁₇ Xaa₁₈ Ile Leu Xaa₂₁ Xaa₂₂wherein Xaa in position 1 is Arg, Lys, Asp or noneXaa in position 2 is Trp, Gly, Lys or Arg,Xaa in position 3 is Ile, Leu, Val or MetXaa in position 4 is Ile, Val or LeuXaa in position 5 Leu, Met, Val or ProXaa in position 12 is Arg, LysXaa in position 13 is Met or Leu,Xaa in position 15 is Ser, Cys or Gln,Xaa in position 17 is Thr, Val, Ile, Ser or Ala,Xaa in position 18 is Ser, Gly or Thr,Xaa in position 21 is Asp, Glu, Cys or Gly,Xaa in position 22 is Gly or none, andn=0, 1, 2 or 3;

(SEQ ID NO: 55) Xaa₁ Xaa₂ Xaa₃ Pro Ile Pro Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁Xaa₁₂ [Gly]_(n) Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁Xaa₂₂ Xaa₂₃ Xaa₂₄wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or noneXaa in position 2 is Asn, Ala or LysXaa in position 3 is Pro, Gln, Gly, Ile or LeuXaa in position 7 is Val or AlaXaa in position 8 is Gly or LysXaa in position 9 is Glu, Asp, Lys, Phe or ThrXaa in position 10 is Ile, Met, Val or LeuXaa in position 11 is Tyr, Leu or noneXaa in position 12 is Ser or noneXaa in position 13 is Arg or noneXaa in position 14 is Asp, Arg, Trp, Ala or noneXaa in position 15 is Ile or noneXaa in position 16 is Tyr or noneXaa in position 17 is Lys or ArgXaa in position 18 is Arg, Lys or AspXaa in position 19 is Trp or GlyXaa in position 20 is Ile, Met, Val, Gln or AlaXaa in position 21 is Ile, Val or AlaXaa in position 22 is Leu, Met or ValXaa in position 23 is Gly or CysXaa in position 24 is Leu or none,n=1, 2 or 3, and

(SEQ ID NO: 61) Xaa₁ Xaa₂ Ile Ile Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Leu Xaa₁₁[Gly]_(n) [Arg]_(m) Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅wherein the Xaa in position 1 is Pro, Lys, Arg or noneXaa in position 2 is Glu, Arg, Phe or LysXaa in position 5 is Pro or ThrXaa in position 6 is Met, Thr or NleuXaa in position 7 is Phe or LeuXaa in position 8 is Ser, Thr, Ala or MetXaa in position 9 is Ala, Glu or LeuXaa in position 11 is Ser or noneXaa in position 12 is Ala, Arg or noneXaa in position 13 is Ile, Leu or noneXaa in position 14 is Ser, Ala, Leu or noneXaa in position 15 is Tyr, Glu or AspXaa in position 16 is Gly or AspXaa in position 17 is Ala or LeuXaa in position 18 is Thr, Ile, Val, Leu or Asn,Xaa in position 19 is Pro, Thr or SerXaa in position 20 is Tyr, Phe, Nleu, His or GlnXaa in position 21 is Asp, Asn, Leu or AlaXaa in position 22 is Leu, Ile, Val or AsnXaa in position 23 is Asn, Tyr, Cys or GlyXaa in position 24 is Thr, Met, Ile, Ala, Val or noneXaa in position 25 is Gly or nonen=1, 2 or 3 and m=0, 1, 2 or 3 independent of each other,the terminal ends of each HIV specific peptide may be free carboxyl- oramino groups, amides, acyls, acetyls or salts thereof.

The HIV-specific peptide sequences have the potential to serve as a goodantigen wherein the antigen comprises at least one peptide selected fromthe group of sequences of SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 55 orSEQ ID NO: 61. The antigenicity may be adapted through adjusting theratio or concentration of different peptides or size of the peptides byfor instance dimerisation or polymerisation and/or immobilisation to asolid phase. The antigen comprises two or more polypeptide sequences,according to the invention, which are either linked by a bridge forinstance a disulphide bridge between the Cys residues of the chains orbridges like C₁-C₈alkylen possibly intervened by one or more heteroatomslike 0, S, or N or preferably they are unlinked. The chains may beimmobilized to a solid phase in monomeric, dimeric or oligomeric forms.Further amino acids may be added to the ends in order to achieve an

arm

to facilitate immobilization.

All amino acids in the HIV-specific peptides of the invention can be inboth D- or L-form, although the naturally occurring L-form is preferred.

The C- and N-terminal ends of the HIV-specific peptide sequences coulddeviate from the natural sequences by modification of the terminalNH₂-group and/or COOH-group, they may for instance be acylated,acetylated, amidated or modified to provide a binding site for a carrieror another molecule.

The HIV-specific peptides according to the invention are consisting of 6to 50 amino acids, preferably between 10 and 30 amino acids. They arecovering all natural variation of amino acids in the identifiedpositions.

The polypeptide antigen according to the invention is either in a freeor in a carrier-bound form. The carrier or solid phase to which thepeptide is optionally bound can be selected from a vide variety of knowncarriers. It should be selected with regard to the intended use of theimmobilized polypeptide as a diagnostic antigen or as an immunizingcomponent in a vaccine.

Examples of carriers that can be used for e.g. diagnostic purposes aremagnetic beads or latex of co-polymers such as styrene-divinyl benzene,hydroxylated styrene-divinyl benzene, polystyrene, carboxylatedpolystyrene, beads of carbon black, non-activated or polystyrene orpolyvinyl chloride activated glass, epoxy-activated porous magneticglass, gelatine or polysaccharide particles or other protein particles,red blood cells, mono- or polyclonal antibodies or fab fragments of suchantibodies.

In a preferred embodiment of the vaccine according to the presentinvention it comprises antigens containing the peptides of the SEQ IDNO: 1, 4, 9 and 15, more preferred the peptides occur in the ratio1:1:1:1.

In a further preferred embodiment the vaccine composition contains theantigens;

(SEQ ID NO: 49) RALGPAATLQTPWTASLGVG-NH₂ (SEQ ID NO: 52)RWLLLGLNPLVGGGRLYSPTSILG-NH₂ (SEQ ID NO: 57)RAIPIPAGTLLSGGGRAIYKRTAILG-NH₂ and (SEQ ID NO: 64)RFIIPNIFTALSGGRRALLYGATPYAIG-NH₂. (NI in position 6 is Norleucine).

One of the sequences contains a B-cell epitope and will activate thehumoral immune system, whereas the other sequences contribute withCTL-epitopes and the amino acid changes implemented within the frame ofthe CTL-epitope are designed to achieve enhanced binding. Other aminoacid changes have been conducted in order to facilitate the synthesis ofthe peptide and/or increase the solubility of the peptide.

Example 1 Synthesis of Peptides Using Conventional Techniques for LinearSequences Preparation of APTKAKRRVVQREKR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of F-moc synthesis(Atherton et al. 1978 3. Chem. Soc. Chem Commun 539), which is belowtermed “the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1822.2

-   -   Experimental molecular weight: 1823.0 ES+

Preparation of APTKAKR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 769.6

-   -   Experimental molecular weight: 760.7 ES+

Preparation of APTRAKR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 797.6

-   -   Experimental molecular weight: 797.6 ES+

Preparation of APTEAKR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 770.9

-   -   Experimental molecular weight: 770.9 ES+

Preparation of RVVEREK

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 914.1

-   -   Experimental molecular weight: 913.9 ES+

Preparation of RVVQREK

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 913.1

-   -   Experimental molecular weight: 913.0 ES+

Preparation of AKRRVV

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 726.9

-   -   Experimental molecular weight: 726.9 ES+

Preparation of DRPEGIEEEGGERDR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1742.1

-   -   Experimental molecular weight: 1742.8

Preparation of VERYLKDQQLLG

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1460.7

-   -   Experimental molecular weight: 1460.1

Preparation of VERYLKDEELLG

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1462.6

-   -   Experimental molecular weight: 1463.0

Preparation of VERYLKDNNLLG

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1432.6

Preparation of QLLLNGSLAEEEIVI

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1639.9

Preparation of QLLLNGSLAEEEVVI

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1625.9

Preparation of QLLLNSLAEEEVVI

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1568.8

Preparation of APTKAKRGGGAPTRAKRGGGAPTEAKR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 2647.0

-   -   Experimental molecular weight: 2646.3 ES+

Preparation of RVVEREKGGGAKRRVVGGGRVVQREK

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 2862.3

-   -   Experimental molecular weight: 2863.3 ES+

Preparation of GGAKRRVVGGAKRRVVGQREKRAV

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 2590.1

Preparation of CGGAKRRVVGGAKRRVVGQREKRAV

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 2693.2

Preparation of GGAKRRVVGGAKRRVVGGQREKR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 2476.9

Preparation of CGGAKRRVVGGAKRRVVGGQREKR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 2580.0

Preparation of GGAKRRVVGGAKRRVV

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1665.0

Preparation of GCGAKRRVVGGAKRRVV

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1768.1

Preparation of GGGDQQLLGGAEEEIVGGIEEEGGERDRDR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 3127.2

Preparation of CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 3230.4

Preparation of GGDQQLLGGAEEEIVGGGERDR

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 2242.4

Preparation of CGGGDQQLLGGAEEEIVGGIEEEGG

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 2402.5

Preparation of GGAEEEVVGGDQQLL

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1499.6

Preparation of CGGAEEEVVGGDQQLL

The peptide was synthesized in amide form, from the correspondingstarting point according to the general description of synthesis.

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 1602.7

Example 2 Synthesis of Complexed Peptides Preparation of

CGGAKRRVVGGAKRRVVGQREKRAV | CGGGDQQLLGGAEEEIVGGIEEEGGERDRD

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 5750.4

-   -   Experimental molecular weight:

Preparation of

CGGAKRRVVGGAKRRVVGGQREKR | CGGGDQQLLGGAEEEIVGGIEEEGG

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 4965.6

-   -   Experimental molecular weight:

Preparation of

 CGGAEEEVVGGDQQLL  | GCGGAKRRVVGGAKRRVV

Purity (HPLC): more than 90%.

Mass spectral analysis: Theoretical molecular weight: 3410.9

-   -   Experimental molecular weight:

Example 3 Recognition of SEQ ID NO: 1 Alone and in Combination with SEQID NOs: 6, 8 and 9 by Pooled Human Sera from HIV Chronically InfectedIndividuals, LTNP and Non-Infected Blood Donors

Seroreactivity to SEQ ID NO: 1 alone or in combination with SEQ ID NOs.6 (with the sequence DRPEGIEEEGGERDR), 8 and 9 was determined accordingto a general ELISA principle either using magnetic particles as a solidsupport or attachment of peptides to a 96-well tray.

Methods:

In the system described below, peptide was coated on to magneticparticles using generally accepted techniques. 300 μg was coated ontoparticles for all peptides with the exception of SEQ1 where 600 μg wasused. SEQ ID NO: 1 (from C5) and SEQ ID NO: 6, 8 and 9 (from gp41) werepreincubated overnight at 4 degrees C. to allow interactions to formbetween between C5 and gp41 sequences respectively and all combined.Sera were then incubated with the peptide coated beads according toestablished protocols. Visualisation of antibody binding to C5 peptideswas achieved using protein G that can bind immunoglobulins fromdifferent species coupled to alkaline phosphatase. The positive controlwas commercially available serum from a sheep immunised with the C5derived sequence APTKAKRRVVQREKR (SEQ ID NO: 1).

Pooled sera from 25 LTNP were tested for seroreactivity to SEQ ID NO: 1alone and SEQ ID NO: 1 when in combination with SEQ ID NOs: 6(DRPEGIEEEGGERDR), 8 and 9 respectively and all combined. Pooled serawere also tested from 12 HIV positive, chronically infected individualsand 20 sera from blood donors. The results are shown in Table A:

TABLE A Results of seroreactivity of pooled sera to SEQ ID NO: 1 and SEQID NO: 1 combined with sequences to gp41. Positivity is determinedvisually. HIV Blood- chronically donors infected LTNP- Pos. Peptide poolpool pool Control APTKAKRRVVQREKR (= SEQ ID NO: 1) =  2+ 4+ >4+ (600μg/ml) APTKAKRRVVQREKE (SEQ ID NO: 1) (−) Neg 3+ 4+ 300 μg/ml +DRPEGIEEEGGERDR (SEQ ID NO: 6) 300 μg/ml + VERYLKDQQLLG (SEQ ID NO: 8)300 μg/ml + VERYLKDEELLG (SEQ ID NO: 9) 300 μg/ml APTKAKRRVVQREKE (SEQID NO: 1) + + 2+ 4+ 300 μg/ml + DRPEGIEEEGGERDR (SEQ ID NO: 6) 300 μg/mlAPTKAKRRVVQREKE (SEQ ID NO: 1) (−) Neg 2+ 4+ 300 μg/ml + VERYLKDQQLLG(SEQ ID NO: 8) 300 μg/ml APTKAKRRVVQREKE (SEQ ID NO: 1) + Neg + 4+ 300μg/ml + VERYLKDEELLG (SEQ ID NO: 9) 300 μg/ml

Results/Discussion Points:

The results in Table A show that pooled LTNP sera generally providestrong reactivity to SEQ ID NO: 1 from HIV-1 when compared to pooledsera from patients chronically infected with HIV—this has been reportedpreviously. However, combining SEQ ID NO: 1 with other peptides derivedfrom gp41 (e.g. all combined or only SEQ ID NO: 8) reduced the level ofbackground observed in blood donors as well as responses in pooled serafrom chronically infected individuals. The response in LTNP remainsstrong.

Example 4 Recognition of SEQ ID NO: 1 and SEQ ID NO: 1 in Combinationwith SEQ ID NO: 6 by Individual Human Sera from HIV ChronicallyInfected, LTNP, Blood Donors

Seroreactivities of individual LTNP patient sera to SEQ ID NO: 1 (16 μg)alone and in combination with SEQ ID NO: 6 (16 μg) were determined usingan ELISA plate as a solid support. Sheep anti-05 antibodies were used asa positive control. Optical density (OD) at 280 nm was used as a readout following the enzymatic reaction from protein G coupled to alkalinephosphatase.

Results have shown that a greater proportion of LTNP sera (n=8, 2 mostright-hand, grey bars) have reactivity to SEQ ID NO: 1 when it is incombination (i.e. >80% ratio) with SEQ ID NO: 6 (DRPEGIEEEGGERDR) whencompared to the reactivity against SEQ ID NO: 1 alone (n=6, mostleft-hand hatched bar, <0% ratio). For LTNP sera that only had a lowresponse to SEQ ID NO: 1, this effect was enhanced when SEQ ID NOs: 1and 6 were combined. This demonstrates that the C5:gp41 complex has theability to capture and increase the response dramatically, even when theresponse from C5 alone is low; Results have shown that the OD was highin the serum samples that only showed binding to SEQ ID NO: 1. However,when combined with the gp41 sequence, the responses to C5 alone werereduced since antibodies now preferably bound the combination.

Data has been made showing the magnitude of responses in individual serafrom LTNP, chronically infected patients and blood donors to SEQ ID NO:1 alone and SEQ ID NO: 1 in combination with SEQ ID NO: 6(DRPEGIEEEGGERDR). There was a greater response to SEQ ID NO: 1 aloneand SEQ ID NO: 1 combined with SEQ ID NO: 6 (DRPEGIEEEGGERDR). amongLTNP patients compared to patients chronically infected with HIV. Themedian OD value for binding to SEQ ID NO: 1 and SEQ ID NO: 6 incombination is higher than binding to SEQ ID NO: 1 alone for both LTNPand patients chronically infected with HIV, showing that combinationwith SEQ ID NO:6 improved seroreactivity. Responses in blood donors areconsistently low, there is a very tight interquartile range and nodifference in seroreactivity to C5 alone or in combination with SEQ IDNO: 6 (DRPEGIEEEGGERDR) in this negative control.

A Wilcoxon rank-test performed on the OD-values derived from SEQ ID NOs:1 and 6 combined on LTNP-sera and the OD-values derived from SEQ ID NOs:1 and 6 combined on HIV-sera, gives that the true median differs withina 25% confidence-interval.

Example 5 Immunological Studies Rabbit Immunizations

New Zealand White female rabbits (n=3) were immunized intradermally atweeks 0, 2 & 6 with 1 ml of BI400-B vaccine consisting of 500 μg BI400-Bin 50% V/V Freund's adjuvant (i.e. Complete Freund's adjuvant used forpriming, followed by boostings with Incomplete Freund's adjuvant).Individual blood serum was isolated for ELISA.

Direct ELISA for Human Sera

50-100 μl of a mixture of BI400-015 and -201 (pre-incubated in Coatingbuffer—0.05M Na₂CO₃ pH9.6; denoted CB—in cold at 16 μg/ml for eachpeptide 1-3 days prior to coating) or just CB (background control) wasused for coating wells in microtiter plates at 4° C. overnight. Themicrotiter plates were then washed 3x with washing buffer (PBS+1% v/vTriton-X100; denoted WB), followed by 2 h blocking at room temperature(RT) with 200 μl/well of blocking buffer (PBS+1% w/v BSA). Plates werethen washed 3x with WB, followed by 1 h incubation at 37° C. with 50-70ul/well of added human (serial dilutions ranging from 1:1-1:250 indilution buffer (PBS+1% v/v Triton-X100+1% w/v BSA; denoted DB)). Plateswere then washed 6x with WB, followed by 1 h incubation at RT with 70μl/well of Alkaline Phosphatase-conjugated Protein G (3 μg/ml in DB;Calbiochem 539305). Plates were then washed 6x with WB, followed by10-60 min incubation at room temperature with 100 μl/well of 0.3% w/v ofPhenophtalein monophosphate (Sigma P-5758). Plates were finally quenchedby adding 100 μl/well of Quench solution (0.1M TRIS+0.1M EDTA+0.5MNaOH+0.01% w/v NaN₃; pH14), followed by ELISA reader (ASYS UVM 340) at550 nm.

Competitive ELISA for Rabbit Sera after Immunization with BI400-B

50-100 μl of a mixture of BI400-015 and -201 (pre-incubated in Coatingbuffer—0.05M Na₂CO₃ pH9.6; denoted CB—in cold at 16 μg/ml for eachpeptide 1-3 days prior to coating) or just CB (background control) wasused for coating wells in microtiter plates at 4° C. overnight. Plateswere then washed 3x with washing buffer (PBS+1% v/v Triton-X100; denotedWB), followed by 2 h blocking at room temperature (RT) with 200 μl/wellof blocking buffer (PBS+1% w/v BSA). Plates were then washed 3x with WB,followed by 1 h incubation at 37° C. with 60-100 μl/well of added rabbitserum samples (diluted 1:10-1:250 final concentration) preincubatedtogether (4° C. overnight) with serial dilutions (ranging from 10-1000μM final concentration) of 400-SEQ.B, BI400-015, BI400-201, BI400-204d,recombinant gp41 (Shin-Won Scientific, SWO 102 gp41), BI301-23(irrelevant protein; control), no peptide (i.e. PBS; control), LTNP-serapools (diluted 1:10 final concentration), or Blood donor sera-pools(diluted 1:10 final concentration; control). Plates were then washed 6xwith WB, followed by 1 h incubation at RT with 70 μl/well of AlkalinePhosphatase-conjugated Goat-anti-Rabbit-Ig (6 μg/ml; Dako D0487). Plateswere then washed 6x with WB, followed by 10-60 min incubation at RT with100 μl/well of 0.3% w/v of Phenoftalein monophosphate (Sigma P-5758).Plates were finally quenched by adding 100 μl/well of Quench solution(0.1M TRIS+0.1M EDTA+0.5M NaOH+0.01% w/v NaN₃; pH14), followed by ELISAreader (ASYS UVM 340) at 550 nm.

Results

Data have been made that demonstrates that sera from rabbits immunizedwith the vaccine antigen 400 SEQ-B bound to peptides corresponding toC5/gp41 (015/201) in the presence of PBS. This binding could beinhibited by recombinant gp41 as well as by peptides derived from C5(015), gp41 (201), and C2 (204d) as well as by 400-SEQ-B itself. Thebinding could not be inhibited using an irrelevant peptide (B301-23).

Anti-05/gp41 sera from BI400-B immunized rabbits is competitivelyinhibited by LTNP-sera pools, but not with BD control sera.

Also, antibodies against C5/gp41 were observed in 26/43 natural virussuppressor HIV patients with viral loads <15000 copies/ml) and in 4/15HIV patients with viral loads above 15000 copies/ml. Furthermore,significantly (p=0.018 when using a Mann-Whitney test) higher anti-05IgG responses (i.e. grouped with respect to OD-value measured at sameserum dilution) were observed in HIV-1 patients with viral load below15000 copies/ml (n=43) compared to patients with viral load above 15000copies/ml (n=15).

To conclude, the results from the immunization studies with BI400-Bdemonstrate that it is possible to generate peptides that elicitantibody responses to C5 and gp41/C2 not only as individual componentsbut also as complexes. The specificity of these antibody responses isconfirmed in blocking studies using specific peptide antigens.Furthermore, antibodies generated to these peptides in animal models arecomparable with antibodies elicited in natural HIV infection andassociated with longterm nonprogression. These results show that thesepeptides are suitable for diagnostics as well as the development of avaccine targeting HIV-induced immune activation. The finding thatBI400-B elicits antibodies that bind to the complex between gp41 and C5,and that these antibodies compete with antisera against the same complexepitopes in LTNP HIV patients indicates that it is possible to stimulateimmune responses against these epitopes and thereby induce an LTNP-likecondition in patients which do not themselves raise antibodies of thistype against HIV.

Example 6

In the following, a summary of the procedures for the synthesis andpurification of C5-Peptide is given. Experience is still limited whichmay eventually lead to improvements in the manufacture and quality ofthis product.

The SPPS synthesis was started with 15 mmoles (A-chain) and 30 mmoles(B-chain) of the resin. After purification of a part of the crudeC5-Peptide, 16.6 g of final product were obtained.

Stage 1: Solid Phase Peptide Synthesis

The amino acid sequences of the A-chain(H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH₂)and the B-chain(H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH₂)are assembled by standard solid phase peptide synthesis (SPPS) using theFmoc-strategy. (W. C. Chan and P. D. White; Solid Phase PeptideSynthesis—A Practical Approach, Oxford University Press Inc., New York,2000, ISBN 978-0-19-963724-9)

The solid phase is transferred into an SPPS reactor and the synthesiscycle is started with the Fmoc-deprotection. Following the deprotectionstep, the peptide chain is elongated by the coupling of the followingN-α-protected AA derivative or the dipeptide according to the amino acidsequence in the presence of suitable activating agents. To avoid theformation of long peptide sequences as by-products, a systemicacetylation of unreacted peptide chains (capping procedure) can beperformed after every coupling step.

For each single step, the solvents and/or reagents are added, and thereaction mixture is stirred and then filtered to remove solvents and/orreagents from the resin. Single steps of the SPPS cycle may be repeatedin case the reaction is incomplete. The SPPS cycle is repeated until thesolid phase carries the complete amino acid sequence of the A-chain orthe B-chain.

For the A-chain, a final Fmoc-deprotection is performed and the SPPS iscompleted by drying the peptide resin under reduced pressure.

The B-chain is further modified with a bromoacetyl linker at Lys². Thisprocedure consists of selectively cleaving the side-chain protectinggroup of Lys² and coupling bromoacetic acid to Lys² in the presence of asuitable activating agent. If the coupling reaction is incomplete,recoupling procedures can be performed. The SPPS is then completed bydrying the peptide resin under reduced pressure.

Stage 2: Cleavage from the Resin Including Cleavage of the Acid LabileProtecting Groups

Cleavage of the peptides from the resin and concomitant cleavage of theacid labile protecting groups is accomplished by treatment with TFA inthe presence of water. Scavengers are added as needed to trap reactivecations and to avoid alkylation of side-chain functions. After filteringoff and washing the resin with TFA, the products are precipitated inIPE. They are filtered off, washed with IPE, and dried under reducedpressure.

Stage 3: Purification of the Intermediates by Preparative HPLC (TFASystem)

The A-chain and the B-chain obtained in the previous stage are purifiedby preparative HPLC on reversed phase columns with ACN gradient elution(TFA system) and UV detection at A=220 nm.

Portions of the peptides are dissolved in water or a mixture of waterand acetic acid and loaded onto the column. Subsequently, the ACNgradient of the TFA system is started. The collected fractions arechecked by analytical HPLC and pooled accordingly.

Side fractions can be repurified with the TFA system. Finally, thepooled fractions with adequate purity are lyophilized.

Stage 4: Coupling of A-Chain and B-Chain

The coupling of the two peptide chains is performed by the addition of asolution of the B-chain (1 equivalent) in aqueous TFA to a solution ofthe A-chain (1 equivalent) in TRIS buffer (adjusted to pH 8.5 by theaddition of hydrochloric acid). Additional TRIS buffer is added tomaintain a pH >8 in the reaction mixture. The reaction mixture is thenstirred and the reaction progress is monitored by analytical HPLC. Uponcompletion, the pH of the reaction mixture is lowered to approx. pH 3 bythe addition of TFA.

Stage 5: Purification by Preparative HPLC (TFA System)

The C5-peptide obtained in the previous stage is purified by preparativeHPLC on reversed phase columns with ACN gradient elution (TFA system)and UV detection at A=220 nm.

Portions of the C5-peptide are directly loaded onto the column.Subsequently, the ACN gradient of the TFA system is started. Thecollected fractions are checked by analytical HPLC and pooledaccordingly.

Side fractions can be repurified with the TFA system. Finally, thepooled fractions with adequate purity are lyophilized.

Stage 6: Ion Exchange, Microfiltration, and Lyophilization

The last stage of the manufacture of C5-Peptide is the ion exchange fromthe TFA salt, obtained in the previous stage, into the acetate salt.

The lyophilized material from the TFA purification is dissolved in 5%acetic acid and the solution loaded onto the ion exchange resin (acetateform). The elution is performed with 5% acetic acid and checked by TLC.The product solution is filtered through a 0.2 μm membrane filter andlyophilized to yield the final product as a white to off-white material.

Example 7 Preparation of HIV-Specific Peptides According to theInvention Description of the Preparation of the Peptides

The peptides of the invention can be produced by any known method ofproducing a linear amino acid sequence, such as recombinant DNAtechniques. A nucleic acid sequence which encodes a peptide of theinvention or a multimer of the said peptides, is introduced into anexpression vector. Suitable expression vectors are for instanceplasmids, cosmids, viruses and YAC (yeast artifical chromosome) whichcomprise necessary control regions for replication and expression. Theexpression vector may be stimulated to expression in a host cell.Suitable host cells are for example bacteria, yeast cells and mammalcells. Such techniques are well known in the art and described forinstance by Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. Otherwell-known techniques are degradation or synthesis by coupling of oneamino acid residue to the next one in liquid phase or preferably on asolid phase (resin) for instance by the so-called Merrifield synthesis.See for instance Barany and Merrifield in the Peptides, Analysis,Synthesis, Biology, Vol. 2, E. Gross and Meinhofer, Ed. (Acad.Press,N.Y., 1980), Kneib-Coronier and Mullen Int. 3. Peptide Protein Res., 30,p.705-739 (1987) and Fields and Noble Int. J. Peptide Protein Res., 35,p.161-214 (1990).

In case a linked or cyclic peptide is desired, the amino acid sequenceis subjected to a chemical oxidation step in order to cyclize or linkthe two cysteine residues within one or between two peptide sequences,when the appropriate linear amino acid sequences are synthesized, seeAkaji et al., Tetrahedron Letter, 33, 8, p.1073-1076, 1992.

General Description of Synthesis

All peptide derivatives prepared in the Examples given below weresynthesized on a Milligen 9050 Peptide Synthesizer using a standardprogram. The resin used was Tenta Gel P RAM with a theoretical loadingof 0.20 meq/g (RAPP POLYMERE GmbH, Tubingen). The final product of thesynthesis was dried in vacuo overnight. The peptide was then cleavedfrom the resin by treatment with 90% trifluoroacetic acid in thepresence of ethandithiol (5%) and water (5%) as scavengers (1.5 hours atRT). Then the resin was filtered and washed on filter with additionaltrifluoroacetic acid (100%) (2×20 ml). The combined filtrates wereevaporated in vacuo (water bath at RT) and the residue was trituratedwith ethyl ether (200 ml) and the precipitated product filtered off. Thesolid was promptly dissolved on filter with glacial acetic acid (100 ml)and added to 1.5 l of 20% acetic acid in methanol and treated with 0.1 Msolution of iodine in methanol until a faint brown colour remained. ThenDowex 1×8 ion exchange in acetate form (15 g) (Bio-Rad, Richmond,Calif.) was added and the mixture filtered. The filtrate was evaporatedand the residue freeze-dried from acetic acid. The product was thenpurified by reversed phase liquid chromatography on a column filled withKromasil® 100-5 C8 (EKA Nobel, Surte, Sweden) in a suitable systemcontaining acetonitrile in 0.1% trifluoroacetic acid water solution. Thesamples collected from the column were analyzed by analytical highperformance liquid chromatography (HPLC) (Beckman System Gold, USA)equipped with a Kromasil® 100-5 C8 Column (EKA Nobel, Surte, Sweden).Fractions containing pure substance were pooled, the solvent wasevaporated and the product freeze-dried from acetic acid. The final HPLCanalysis was performed on final product, and the structure of thepeptide was confirmed by amino acid analysis and mass spectrometry(LDI-MS).

All amino acids used during the synthesis were L-amino acids and theywere protected with a fluorenylmethoxy-carbonyl group at the α-aminofunction. The side chains were protected as follows:

Cys (Trt), Gln(Trt), Glu(OtBu), Thr(tBu).

The abbreviations, within the brackets are:

Trt=triphenylmethylt-Bu=tert. ButylOtBu=tert. Butylester

The amino acid derivatives was supplied by Bachem AG, Switzerland.

Example 8 Preparation of K A L G P G A T L Q T P W T A C Q G V G-NH₂(SEQID NO: 48)

The peptide was synthesized in amide form, from corresponding startingmaterials according to the general description of synthesis. The puritywas determined by HPLC analysis and the structure was confirmed by aminoacid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): 87%

Example 9 Preparation of R A L G P A A T L Q T P W T A S L G V G (SEQ IDNO: 49)

The peptide was synthesized in amide form, from corresponding startingmaterials according to the general description of synthesis. The puritywas determined by HPLC analysis and the structure was confirmed by aminoacid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): more than 95%

Molecular weight (free base): 1966

Molecular formula: C₈₈H₁₄₄O₂₅N₂₆

Example 10 Preparation of W I I P G L N P L V G G G K L Y S P T S I L CG-NH₂(SEQ ID NO: 51)

The peptide was synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis.The purity was determined by HPLC analysis and the structure wasconfirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): 95%

Mass spectral analysis: Theoretical molecular weight: 2454.9

Experimental molecular weight: 2454.8 ES+

Example 11 Preparation of R W L L L G L N P L V G G G R L Y S P T S I LG (SEQ ID NO: 52)

The peptide was synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis.The purity was determined by HPLC analysis and the structure wasconfirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): more than 95%

Molecular weight (free base): 2552

Molecular formula: C₁₁₉H₁₉₅O₂₉N₃₃

Example 12 Preparation of K I L L G L N P L V G G G R L Y S P T S I L G(SEQ ID NO: 53), R L L L G L N P L V G G G R L Y S P T T I L G (SEQ IDNO: 54) and N I P I P V G D I Y G G G D I Y K R W Q A L C L (SEQ ID NO:70)

The peptides are synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis.The purity are determined by HPLC analysis and the structures areconfirmed by amino acid analysis and mass spectrometry (LDI-MS).

Example 13 Preparation of R N I P I P V G D I Y G G G D I Y K R W Q A LC L (SEQ ID NO: 56)

The peptide was synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis.The purity was determined by HPLC analysis and the structure wasconfirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): 85%

Mass spectral analysis: Theoretical molecular weight: 2817.3

Experimental molecular weight: 2813.7 ES+

Example 14 Preparation of R A I P I P A G T L L S G G G R A I Y K R W AI L G (SEQ ID NO:57)

The peptide was synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis.The purity was determined by HPLC analysis and the structure wasconfirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): more than 95%

Molecular weight (free base): 2707

Molecular formula: C₁₂₅H₂₀₈O₂₉N₃₈

Example 15 Preparation of A L P I P A G F I Y G G G R I Y K R W Q A L G(SEQ ID NO: 58), K I P I P V G F I G G G W I Y K R W A I L G (SEQ ID NO:59) and K I P I P V G T L L S G G G R I Y K R W A I L G (SEQ ID NO: 60)

The peptides are synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis.The purity are determined by HPLC analysis and the structures areconfirmed by amino acid analysis and mass spectrometry (LDI-MS).

Example 16 Preparation of K F I I P N I F S A L G G A I S Y D L N T N IL N C I (SEQ ID NO: 62)

The peptide was synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis. NIin the sequence is Norleucine. The purity was determined by HPLCanalysis and the structure was confirmed by amino acid analysis and massspectrometry (LDI-MS).

Purity (HPLC): more than 80%

Mass spectral analysis: Theoretical molecular weight: 2783.3

Experimental molecular weight: 2783.3 ES+

Example 17 Preparation of K F I I P N I F S A L S G G G A I S Y D L N TF L N C I G (SEQ ID NO: 63)

The peptide was synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis. NIin the sequence is Norleucine. The purity was determined by HPLCanalysis and the structure was confirmed by amino acid analysis and massspectrometry (LDI-MS).

Purity (HPLC): more than 80%

Mass spectral analysis: Theoretical molecular weight: 2932.4

Experimental molecular weight: 2931.8 ES+

Example 18 Preparation of R F I I P N I F T A L S G G R R A L L Y G A TP Y A I G (SEQ ID NO:64)

The peptide was synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis. NIin the sequence is Norleucine. The purity was determined by HPLCanalysis and the structure was confirmed by amino acid analysis and massspectrometry (LDI-MS).

Purity (HPLC): more than 95%

Molecular weight (free base): 2894

Molecular formula: C₁₃₇H₂₁₇O₃₂N₃₇

Example 19 Preparation of K I I P N I F S A L G G G R L L Y G A T P Y AI G (SEQ ID NO: 65), R I I P N I F T A L S G G G R L L Y G A T P Y A I G(SEQ ID NO: 66) and W I I P N I F S A L G G A I S Y D L N T N I L N C I(SEQ ID NO: 71)

The peptides are synthesized in amide form, from the correspondingstarting materials according to the general description of synthesis.The purity are determined by HPLC analysis and the structures areconfirmed by amino acid analysis and mass spectrometry (LDI-MS).

Example 20 Dimerisation Via Disulphide Bridge

The peptide sequences of the Examples 8 and 10 were linked via anoxidation step to form a dipeptide wherein the cysteine residues formeda disulphide bridge. The bridge was formed in either ways;

A) Oxidation with I₂ Equal amounts of the peptides were dissolved inacetic acid/methanol (1:4) and 0.1 M I₂ in methanol was added yielding amixture of the dimer.orB) Oxidation via [Cys(Spy)¹⁶]-SEQ ID NO:48. 2.3 mM of the peptide of SEQID NO:48 dissolved in 2 M AcOH (aq) and 2-propanol (1:1) was treatedwith 2,2 dithiodipyridin (3 eqv) to yield [Cys(Spy)¹⁶]-SEQ ID NO:48.Equal amounts of [Cys(Spy)¹⁶]-SEQ ID NO:48 and peptide of SEQ ID NO:51were dissolved in 10 mM NH₄Oac (aq pH=6, 5) and methanol (5:2) to yieldthe dimer of SEQ ID NO:67.

The purity of the peptide was determined by HPLC analysis and thepeptide structure was confirmed by amino acid analysis. The peptidecontent (aminoacid free base) was 80%,

Purity (HPLC): 92%.

Example 21

A vaccine comprising the peptides of the SEQ ID NO: 49, 52, 57 and 64was prepared. The freeze-dried peptides were dissolved in sterile waterat a final concentration of 4 mg/ml. The final salt concentration was0.9%. A preparation of a granulocyte-macrophage-colony stimulatingfactor (GM-CSF) was also prepared, according to the manufacturersdirections for use, to a final concentration of 0.3 mg/ml. The twosolutions are administered intracutaneously. A typical injection dose is100 μl.

Example 22

An antigen solution or suspension is mixed with equal parts of Freund'sadjuvant of Behring, complete or incomplete, and is then finelyemulsified by being drawn up into, and vigurously pressed out of, aninjection syringe, or with a homogenator. The emulsion should remainstable for at least 30 minutes. The antigen-adjuvant emulsions is bestinjected subcutaneously as a depot.

Example 23

Toxicity studies were performed in mice and rats on the peptidecomposition of the vaccine in Example 22. The mouse was selected for thestudy to provide comparative data from a second commonly used rodentspecies. The test substance was a mixture of four peptides supplied asone vial containing lyophilised material for reconstitution withphysiological saline, and dose levels were expressed in terms of totalpeptide load. The individual peptides was present in ratio 1:1:1:1 w/wgiving dose levels of each peptide of 0.0075 mg/kg body weight, 0.075mg/kg body weight and 0.75 mg/kg body weight, which are up to 500 foldthe intended human dose. The test animals were divided into four groupsof ten animals each (five males and five females); a saline controlgroup and groups for low, intermediate and high doses. The testcomposition was administered once, by intravenous infusion into a tailvein at a dose rate of 3 ml/minute. The animals were killed at day 15and 16 by intraperitoneal injection of sodium pentobarbitone.

The results of these studies indicated that the dose levels administeredto the mice and rats elicited no adverse reactions and that the noeffect level was in excess of 3 mg/kg.

Example 24 Immunoassay for Detection of Antibodies Induced by HIV-1

The magnetic particle reagents are to be prepared according to themanufacturers recommended protocol. Dynal AS, is the manufacturer of theDynabeads, which are employed. The magnetic particles coated with ligandare called Reagent 1. A peptide according to the invention is covalentlycoupled to the pre-activated surface of the magnetic particles. It isalso possible to physically absorb the peptide to the surface of themagnetic particles. The concentration of particles in Reagent 1 iswithin the range from 1 mg/ml to 15 mg/ml. The particle size variesbetween 0.2 μm to 15 μm. The concentration of peptides is within therange from 0.01 mg/mg particle to 1 mg/mg particle.

The anti human Ig Alkaline Phosphatase (AP) conjugated antibody reagentis prepared according to the recommended protocol of Dako AS. Thisprotocol is a standard procedure in this field. This reagent is calledReagent 2.

The substrate solution phenolphtalein-monophosphate is to be preparedaccording to the recommended protocol of Fluka AG. This protocol is astandard procedure in this field. The substrate solution is calledReagent 3.

The washing and incubation buffer which is used is standard 0.05Mtris-base buffer with the following additional compounds; Tween 20(0.01% to 0.1%), glycerol (0.1% to 10%) and sodium chloride (0.2% to0.1%).

The assay procedure comprises an incubation step wherein 1 drop ofReagent 1 is mixed with 2 drops of washing buffer in each well. Aftermixing, 30 μl of sample is added and the solution is incubated for 5minutes. The magnetic particles can be trapped by a magnet and theliquid removed, before the magnet is separated. Then the wells arewashed twice in 4 drops of washing solution, before incubation withReagent 2. 1 drop of Reagent 2 is added with 2 drops of washing bufferand the solution is incubated for 5 minutes. The magnetic particles canbe trapped by a magnet and the liquid removed, before the magnet isseparated. Then the washing step is repeated before incubation withReagent 3. 2 drops of Reagent 3 is added to each well and the solutionis incubated for 3 minutes. The results can be read against a whitebackground. Positive results are red (3+=strong red) whereas negativeresults are clearly light yellow/brown solutions as obtained in thenegative control.

The immunoassay kit could be used in detection of antibodies, inducedeither by HIV virus or HIV-specific peptides or proteins, for instancethe peptides of the present invention.

The above Examples are only meant as illustrating the invention. It mustbe understood that a person skilled in the art can modify the peptides,antigens and vaccines herein described without deviating from theconcept and scope of this invention as set forth in the claims.

The polypeptides of the invention can be used in a combination of atleast one peptide selected from each group of sequences, SEQ ID NOs: 47,50, 55 and 61 to form antigens and the the active principle of aprophylactic or therapeutic vaccine intended to provide protectionagainst the human immunodeficiency virus type 1 (HIV-1). The vaccine mayinclude compounds having beneficial effects in protecting or stimulatingthe host's immune system (human being or vertebrate animal) for instanceinterleukins, interferons, granulocyte macrophage growth factors,haematopoietic growth factors or similar. Preferably the vaccinecomposition further contain an adjuvant or vehicle, more preferably theadjuvant or vehicle is Monophosphoryl Lipid A (MPL®) possibly with alum,Freund's adjuvant (complete or incomplete) or aluminum hydroxide. Theoptimal amount of adjuvant/vehicle will depend on the type(s) which ischosen.

The peptide or vaccine formulation can be freeze-dried prior to storage.The vaccine may be stored preferably at low temperature, in ampoulescontaining one or more dosage units, ready for use. A typical dosageunit of the peptide according to the invention is within theconcentration range: 1 μg-1 mg per kg bodyweight, preferably within 2μg-0.15 mg per kg body weight. Persons skilled in the art willappreciate that a suitable dose will depend on the body weight of thepatient, the type of disease, severity of condition, administrationroute and several other factors. The vaccine might be administered up totwelve times and through injection, typically it will be administeredabout three times. In preparation of an injection solution the peptidesare dissolved in sterile sodium chloride solution at a finalconcentration of 1 mg/ml per peptide and 0.9% sodium chloride. Typicallyan injection volume is 100 μl to 200 μl (2×100 μl). The peptide ispreferably co-administered with a suitable adjuvant and/or agranulocyte-macrophage growth factor for instance Leucomax® <<SheringPlough>>. Suitable administration may be intracutane, subcutane,intravenous, peroral, intramuscular, intranasal, mucosal or any othersuitable route. Booster administrations may be required in order tomaintain protection.

Example 25

The anti-HIV p24 immune response resulting from Vacc-4x immunizationcould in combination with ART potentially improve immune reconstitutionin patients who have not fully regained a healthy CD4 level(>600×10⁶/L). Potential benefits of Vacc-4x in subjects with incompleteimmune reconstitution include a possible sustained improvement in theimmune response to p24 and HIV.

Potential risks include the discomfort and inconvenience associated withthe immunizations and the risk of known or unknown side effects ofexposure to Vacc-4x and Leukine (rhu-GM-CSF) including, most commonly,local reactions at the site of injections and fatigue (likelihood notyet determined).

The results of non-clinical single-dose studies in mice and ratsindicate that the dose levels of intravenous Vacc-4x elicited no adversereactions and that the no effect level was in excess of 3 mg/kg.

In a rabbit study the effect of Vacc-4x was evaluated in the presence ofconcomitant GM-CSF, the adjuvant used in the clinical program. Localintradermal reactions such as erythema and edema were noted, however,similar effects were noted in control animals both macroscopically andhistological. These local reactions were slightly more pronounced in theVacc-4x treated animals. There were no systemic reactions in this study.These data indicate that Vacc-4x has no limiting toxicology in a modelthat is relevant to the proposed clinical study.

The therapeutic vaccine candidate Vacc-4x, has been studies in a Phase Iand three Phase II clinical studies. The Phase I study enrolled 11HIV-positive subjects, including nine subjects on ART. Subjects weremaintained on ART (if entered on ART); all subjects were treated with 12immunizations of Vacc-4x at a dose of 0.4 mg/injection over a period of26 weeks. Immunizations were performed following injection of rhu-GM-CSF(Leucomax® [molgramostim]) as adjuvant. All subjects experienced one ormore adverse events (AEs); nine subjects experienced events judgedrelated to treatment. The adverse reactions reported were mild ormoderate in severity except for severe local reactions in one subject.No subjects were withdrawn due to treatment-related AEs or toxicologicalreactions; no serious adverse events (SAEs) occurred. Treatment relatedevents observed in more than one subject included painful injection(seven subjects), fatigue-vertigo (four subjects), influenza-likesymptoms (two subjects), and irritated skin at injection site (twosubjects).

All subjects experienced a cell-mediated immune response, measured bydelayed-type hypersensitivity (DTH) skin reaction. Some cell-mediatedimmune response, measured by γ IFN release using enzyme-linkedimmunosorbent spot assay (ELISPOT), was reported for 45% of thesubjects; no antibody response to Vacc-4x peptides was observed.

The Phase II dose-finding study (CTN B-HIV 2/2001) enrolled 40 HIVpositive subjects, of which 38 completed the trial. Subjects weremaintained on ART and treated with 10 immunizations at a dose of 0.4 mg(20 subjects) or 1.2 mg (20 subjects) per Vacc 4x injection, over aperiod of 26 weeks. Immunizations with Vacc 4x were performed followinginjection of rhu-GM-CSF (Leucomax [molgramostim]) as a local adjuvant.ART was interrupted from Week 26 to Week 30 to allow exposure to thesubject's own virus (autologous immunization). ART was resumed from Week30 to Week 38 to allow maturation of immune responses to the Vacc 4xpeptides and to the subject's own virus. ART was discontinued from Week38 to Week 52 when the study was formally concluded. Treatment-relatedAEs were observed in 20 subjects (8 subjects in the 0.4 mg group and 12subjects in the 1.2 mg group). No SAEs were reported during the periodof immunization. One subject experienced a transient vasovagal reactionin conjunction with immunization and the DTH test at Week 26 and Week38. A second subject experienced a vasovagal reaction in conjunctionwith the DTH test at Week 52. For the laboratory parameters, vitalsigns, and performance status, no changes attributable to immunizationwere observed. Changes in HIV RNA, CD4 cell counts, and CD8 cell countsshowed no safety concerns related to immunization.

Immunological responses reported as DTH positive reactions were observedfor all subjects. Overall, positive responses both for induration anderythema were statistically significantly higher in the high dose (HD,1.2 mg Vacc-4x) group compared to the low dose (LD, 0.4 mg Vacc 4x)group. The dose-dependent differences in DTH reactions were maintainedthroughout the study. T-cell proliferation appeared stable after Week 12and demonstrated an HD advantage, consistent with the DTH results. ARTwas interrupted at Week 38 with planned restart when CD4 counts fell toless than 200/μL or when AIDS- or HIV related events were observed (i.e.clinical practice). DTH responses to Vacc-4x (high versus low responsedetermined at Week 38) were associated with reduced viral loads andcorrespondingly improved CD4 counts at the end of the study (Week 52).

During the immunization period, CD4 counts were stable or increased.Interruption of ART resulted in reduction of CD4 counts. However, 14weeks after the last interruption of ART (Week 52), the mean CD4 countswere still above 200×106 cells/L. No difference between the LD and theHD groups was observed. The majority of subjects remained off ARTfollowing completion of the study (Week 52); permission was given tofollow the subjects until they resumed ART. The duration of treatmentinterruption was linked to immune responsiveness to the peptides. Whensubjects were compared to similar subjects in the Netherlands that hadstopped treatment without Vacc-4x administration, a significantly slowerdecline in CD4 cells was noted for the Vacc-4x subjects. The mediantreatment interruption achieved for all the subjects that participatedin the Vacc-4x Phase II clinical study was 31 months.

CTN BI Vacc-4x/2009/1 was an open-label follow-up of study CTNB-HIV-2/2001 to determine whether a re-boost with Vacc-4x couldreactivate or increase the immune response obtained during theimmunization performed in the CTN B-HIV-2/2001 study. The secondaryobjectives were to evaluate: the in vivo immunogenicity of Vacc-4x byevaluation of DTH and to compare the DTH response to DTH in the initialstudy; the effect of Vacc-4x on CD4 counts, CD8 counts and HIV viralRNA; and the safety and tolerability of Vacc-4x. All 26 subjectsincluded in the study received two booster administrations of Vacc-4x.

A total of 74 AEs were reported by 23 subjects. Most adverse events(n=60) were scored as possibly/probably related to the study treatment.The majority (98%) of the related adverse events were mild. Two adverseevents related to study treatment, one headache and one injection siteindurations, were scored as moderate intensity. Itching (injection sitepruritus) was the most frequent reported adverse event related to thestudy treatment. Nineteen patients (73%) reported this adverse event atleast once. Ten of these patients reported itching related to bothimmunizations, while for the other nine patients it was only reportedonce. Five patients reported swelling related to the immunization. Forthree of these patients swelling was reported after both immunizations.No patient died during the study. No patient reported serious adverseevents and no clinically relevant changes were recorded.

The study demonstrated that Vacc-4x peptides induced T cell responseslasting up to seven years. By re-boosting it was possible to increasekilling markers, this again indicates that T cells had increased theirpotential to kill HIV-infected cells. Before re-boosting, all thepatients had returned to CD4, CD8 and viral load levels that weresimilar to those before ART was stopped in the main study. Re-boostinghad no negative effect on the CD4, CD8 and viral load of the patients.No safety concern was reported as a result of the re-boost of thesepatients.

The Phase II Study CT-BI Vacc-4x 2007/1 (EudraCT Number 2007-006302-13)was performed in US and Europe (UK, Germany, Spain and Italy). The studywas a randomized, double-blind, multicenter, immunogenicity study ofVacc-4x versus placebo in patients infected with HIV-1 who havemaintained an adequate response to ART. The primary objective was toevaluate the effect of Vacc-4x immunizations versus placebo on CD4counts, T-cell function (ELISPOT, T-cell proliferative responses andintracellular cytokine staining) and the response to interruption ofART. The necessity to resume ART between the interruption of ART at Week28 and the end of the study at Week 52, due to decreased CD4 count orincreased viral loads, was monitored as one of the primary efficacyendpoints.

In the ITT analysis population, it was concluded that Vacc-4x did notreduce the proportion of subjects requiring resumption of ART after ARTcessation at Week 28 in comparison with placebo. There was also noeffect compared with placebo on the percentage change in CD4 countbetween Week 28 and the last CD4 assessment before resumption of ART.The time to restarting ART was similar in Vacc-4x and placebo-treatedsubjects.

The viral load results after ART cessation varied between subjects withevidence of favourable effects of Vacc-4x immunization over placebo.There were no significant differences in the repeated measures ANOVA forviral load over Weeks 4 to 52 when data included all evaluable subjects,irrespective of whether they were or were not taking ART. In thesubgroup of subjects who remained off ART until Week 52, the averageviral load was lower in the Vacc-4x-treated subjects than the placebogroup. A post-hoc analysis showed the Week 52 (Last Observation CarriedForward [LOCF]) viral load to be statistically significantly lower inthe Vacc-4x group than the placebo group.

The analysis of change in HIV-1 RNA from Week 28 through to Week 52revealed a statistically significant difference between groups in favourof Vacc-4x. The AUC in those who remained off ART at Week 52 was lowerin the Vacc-4x group than in the placebo group. A post-hoc analysisshowed this difference in AUC to be statistically significant.

No safety concern was raised during this study. The study was supervisedby a Data Safety Monitoring Board (DSMB).

Example 26 Test of Peptides Together with IMiDs for IncreasedProliferation, Polyfunctionality, IL-2 Secretion and IFN-γ Production

Expansion of polyfunctional HIV-specific T-cells upon stimulation withDendritic Cells, pre-incubated with peptides to be used according to theinvention, may be studied by methods described by Keersmaecker et al.(J. Virol., 2012 86:9351-9360) and referenced therein, HIV proteins Gagor Nef, they are incubated with peptides to be used according to theinvention, before they are used to stimulate T-cells in a co-culture.

Keersmaecker et al. found that the presence of IMiDs (Lenalidomide(IMiD3; CC-5013) and pomalidomide (IMiD1; CC-4047) during in vitroT-cell stimulation with dendritic cells presenting Gag- or Nef-specificpeptides, resulted in a number of improvements in the function of theT-cells. Among these were; polyfunctional HIV specific CD8+ T cells withenhanced lytic capacity, more Gag antigen epitopes recognized and atlower antigen peptide concentrations, reduced proliferation of CD4+ Tcells with increased number of polyfunctional CD4+ T-cells, increasedIL-2 production by CD8 T-cells, detectable IFN-γ production by CD8+T-cells and CD4 T-cells after antigen stimulation.

-   “Expansion of Polyfunctional HIV-Specific T Cells upon Stimulation    with mRNA Electroporated Dendritic Cells in the Presence of    Immunomodulatory Drugs”-   Brenda De Keersmaecker, Sabine D. Allard, Patrick Lacor, Rik Schots,    Kris Thielemans, and Joeri L. Aerts-   J. Virol. September 2012 86:9351-9360; published ahead of print 20    Jun. 2012, doi:10.1128/JVI.00472-12

Example 27 Suggested Clinical Study Protocol for the Test of PeptideComposition Comprising 4 Peptides in Combination with Lenalidomide andHDAC Inhibitor

Immunizations (four primary immunizations and two booster immunizations)at Weeks 1, 2, 3 and 4, and booster immunizations at Weeks 12 and 13with either:

1) Peptide composition with GM-CSF as adjuvant and Lenalidomide(CC-5013), or

2) Peptide composition with GM-CSF as adjuvant and Placebo forLenalidomide (CC-5013).

3) Placebo

Suggested Doses:

Peptide composition: 0.6, 0.9, 1.2 and 1.5 mg (Equal amount of eachpeptide, ratio of 1:1:1:1 w/w)

Lenalidomide: 5.10, and 25 mg.

Subjects randomized to the Lenalidomide (CC-5013) arm will take a singleoral dose of Lenalidomide (CC-5013) daily the two preceding days beforeimmunization with the Peptide composition and on the day of eachimmunization.

The Peptide composition used according to this clinical trial setupconsists of SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:57, and SEQ ID NO:64.

At week 20 subjects in all study arms will receive 20 mg panobinostat(LBH589) orally on days 1, 3, and 5 (i.e. 3 times a week) every otherweek for a period of 8 weeks (up to week 28) while maintainingbackground ART. This will be followed by a 24 week follow up period (upto week 52). Upon completion of the study, subjects may be invited toparticipate in an additional observational study in which ART will beinterrupted to evaluate the effect of study treatment on virologicalcontrol. Enrolment into this part of the study will be optional anddetermined by the effect of study treatments on the latent HIV-1reservoir. (Maximum duration of treatment interruption: 16 weeks).

In Summary:

Study arm 1: Peptide composition+IMiD+HDAC (panobinostat)Study arm 2: Peptide composition+HDAC (panobinostat)Study arm 3: HDAC (panobinostat)

Depletion of the viral reservoir as a result of the combinationtreatments according to the present invention may be quantified by forinstance following the procedures set forth in Lehrman et al. (TheLancet (366), 2005, pp. 549-555) and references there in. In brief, thisincludes measuring in samples of patient blood obtained before, duringand after treatment; p24 expression from stimulated latently infectedcells, plasma HIV RNA concentration (viral load), and integrated HIV DNAby realtime PCR analysis.

Example 28 Dc/T-Cell Proliferation Assay

Dendritic cells (DC) were generated from monocytes isolated from buffycoat preparations from healthy blood donors. Briefly, peripheral bloodmononuclear cells were separated by a density gradient centrifugationand the monocytes were then negatively isolated using the DynabeadsUntouched Human Monocytes (Invitrogen, Carlsbad, Calif.) following themanufacturer's instructions. The monocytes were cultured with IL-4 (20ng/ml; Immunotools, Friesoythe; Germany) and GM-CSF (100 ng/ml;Immunotools) in X-VIVO15 medium (Lonza, Basel, Switerland) for 5-6 daysto generate immature DC. Cytokines were replenished every 2-3 days. Thematuration of the cells was performed for 24 hours with IFN-γ (1000IU/ml), TNF-α (50 ng/ml), IL-1β (25 ng/ml) IFN-α (3000 IU/ml). Aftermaturation, the DC were pulsed for 2 hours at 37° C. with peptides at 10μg/ml, before extensive washing and co-culture with Peripheral bloodmononuclear cells (PBMC) labelled with a fluorescent dye (VPD450, BDbiosciences, Sam Jose, Calif.). Various ratios with DC:T cell weretested alongside with appropriate controls. IL-2 (50 U/ml) and IL-7 (50ng/mL) (Both, Immunotools) and wells with or without IMiDs were added atthe start of co-culture. At day 6-10, the level of T cell proliferationwas analysed by flow cytometry. The supernatants from the co-culturewells were investigated with Luminex technology to establish anysuppressor activity.

Example 29

The peptides according to the invention used in the following exampleswere synthesized by Schafer-N as c-terminal amides using theFmoc-strategy of Sheppard, (1978) J. Chem. Soc., Chem. Commun., 539.

Cell Penetration Assay

Intracellular Staining for Biotinylated Peptides

96-well U-bottom polystyrene plates (NUNC, cat no: 163320) were used forstaining of human PBMCs. Briefly, 8 ul of N- or C-terminallybiotinylated peptides according to the invention (i.e. 5 mM, 2.5 mM &1.25 mM tested for each peptide) were incubated at 37° C. for 2 h with40 ul of PBMC (12.5×106 cells/ml) from blood donors. Cells were thenwashed 3x with 150 ul of Cellwash (BD, cat no: 349524), followed byresuspension of each cell pellet with 100 ul of Trypsin-EDTA (Sigma, catno: T4424), then incubated at 37° C. for 5 min. Trypsinated cells werethen washed 3x with 150 ul of Cellwash (BD, cat no: 349524), followed byresuspension with BD Cytofix/Cytoperm™ plus (BD, cat no: 554715), thenincubated at 4° C. for 20 min according to manufacturer. Cells were thenwashed 2x with 150 ul PermWash (BD, cat no: 554715). Cells were thenstained with Streptavidin-APC (BD, cat no: 554067) & Anti-hCD11c(eBioscience, cat no: 12-0116) according to manufacturer at 4° C. for 30min aiming to visualize biotinylated peptides & dendritic cells,respectively. Cells were then washed 3x with 150 ul PermWash, followedby resuspension in staining buffer (BD, cat no: 554656) before flowcytometry. Dendritic cells were gated as CD11c+ events outsidelymphocyte region (i.e. higher FSC & SSC signals than lymphocytes). 200000 total cells were acquired on a FACSCanto II flow cytometer with HTSloader, and histograms for both total cells & dendritic cells withrespect to peptide-fluorescence (i.e. GeoMean) were prepared.

Extracellular Staining for Biotinylated Peptides

96-well U-bottom polystyrene plates (NUNC, cat no: 163320) were used forstaining of human PBMCs. Briefly, 8 ul of N- or C-terminallybiotinylated peptides according to table 1 or table 2 (i.e. 5 mM, 2.5 mM& 1.25 mM tested for each peptide; all peptides manufactured by solidphase synthesis by commercial suppliers) were incubated at 37° C. for 2h with 40 ul of PBMC (12.5×106 cells/ml) from blood donors. Cells werethen washed 3x with 150 ul of Cellwash (BD, cat no: 349524), thenstained with Streptavidin-APC (BD, cat no: 554067) & Anti-hCD11c(eBioscience, cat no: 12-0116) according to manufacturer at 4° C. for 30min aiming to visualize biotinylated peptides & dendritic cells,respectively. Cells were then washed 3x with 150 ul of Cellwash (BD, catno: 349524), followed by resuspension in staining buffer (BD, cat no:554656) before flow cytometry. Dendritic cells were gated as CD11c+events outside lymphocyte region (i.e. higher FSC & SSC signals thanlymphocytes). 200 000 total cells were acquired on a FACSCanto II flowcytometer with HTS loader, and histograms for both total cells &dendritic cells with respect to peptide-fluorescence (i.e. GeoMean) wereprepared.

It was clearly seen that the CMI peptides according to the invention hadimproved ability to enter the cell compared to its native counterparts

The data are geomean-value of each testet peptide, as calculated by theFACS Duva software. The Geomean values bytrypsinating/Cytofix/Cytoperm.:

Example 30 Positive CTL Response May Alternatively be Assayed by ELISPOTAssay

Human IFN-gamma cytotoxic T-cell (CTL) response by ELISPOT assayBriefly, at day 1, PBMC samples from HCV patients were incubated inflasks (430 000 PBMCs/cm2) for 2 h at 37° C., 5% CO2 in covering amountof culture media (RPMI 1640 Fisher Scientific; Cat No. PAAE15-039supplemented with L-Glutamine, (MedProbe Cat. No. 13E17-605E, 10% FoetalBovine serum (FBS), Fisher Scientific Cat. No. A15-101) andPenicillin/Streptomycin, (Fisher Acientific Cat. No. P11-010) in orderto allow adherence of monocytes. Non-adherent cells were isolated,washed, and frozen in 10% V/V DMSO in FBS until further usage. Adherentcells were carefully washed with culture media, followed by incubationat 37° C. until day 3 in culture media containing 2 μg/ml finalconcentration of hrGM-CSF (Xiamen amoytop biotech co, cat no:3004.9090.90) & 1 μg/ml hrIL-4 (Invitrogen, Cat no: PHC0043) andoptionally an immunomodulationg agent (IMiD), and this procedure wasthen repeated at day 6. At day 7, cultured dendritic cells (5 000-10 000per well) were added to ELISPOT (Millipore multiscreen HTS) platescoated with 0.5 μg/well anti-human γ Interferon together with thawedautologous non-adherent cells (200 000 per well), antigen samples (1-8ug/ml final concentration for peptide antigens; 5 ug/ml finalconcentration for Concanavalin A (Sigma, Cat no: C7275) or PHA (Sigma,Cat no: L2769)) & anti-Anergy antibodies (0.03-0.05 ug/ml finalconcentration for both anti-PD-1 (eBioscience, cat no: 16-9989-82) &anti-PD-L1 (eBioscience, cat no: 16-5983-82)). Plates were incubatedovernight and spots were developed according to manufacturer. Spots wereread on ELISPOT reader (CTL-ImmunoSpot® S5 UV Analyzer).

Example 31 ELISPOT Assay

At day one, PBMC samples from blood donors were thawed, washed with warmmedium and incubated in flasks (250000PBMCs/cm2) for 24 hours at 37° C.,5% CO2 in covering amount of culture media (RPMI 1640 withultra-glutamine, Lonza, BE12-702F701; 10% Foetal Bovine serum (FBS),Fisher Scientific Cat. No. A15-101; Penicillin/Streptomycin, FisherScientific Cat. No. P11-010) to allow the cells to recover afterthawing. At day two, the cells were added to a Falcon Microtest TissueCulture plate, 96 well flat bottom, at 500 000 cells per well in avolume of 200 μl total medium. Parallel wells were added the indicatedstimuli in duplicate and optionally an immunomodulationg agent (IMiD),or left with medium as a control for 6 days at 37° C., 5% CO₂. After thesix days of incubation, 100 μl of the cell suspension were transferredto an ELISPOT (Millipore multiscreen HTS) plate coated with 1 μg/mlnative influenza M2e protein. After a 24 hour incubation, the plate waswashed four times with PBS+0.05% Tween20, and a fifth time with PBS, 200μl/well. A mouse Anti-human IgG or IgM biotin (Southern Biotech 9040-08and 9020-08) was diluted in PBS with 0.5% FBS and incubated for 90minutes at 37° C. The washing was repeated as described, before 80 μlStreptavidin-Alkaline-Phosphatase (Sigma Aldrich, S289) was added eachwell and incubated at 60 minutes in the dark, at room temperature. Thewells were then washed 2 times with PBS+0.05% Tween20 and 4 times withPBS, 200 μl/well, before the substrate, Vector Blue Alkaline PhosphataseSubstrate kit III (Vector Blue, SK-5300) was added and let to developfor 7 minutes at room temperature. The reaction was stopped with runningwater, the plates let dry and the sport enumerated by an ELISPOT reader(CTL-ImmunoSpot® S5 UV Analyzer).

ELISA

100 μl of antigen as indicated (pre-incubated in Coating buffer—0.05MNa₂CO₃ pH9.6; denoted CB—in cold at 8 μg/ml 1-3 days) or just CB(background control) was used for coating wells in microtiter plates at4° C. The microtiter plates are then washed 3x with washing buffer(PBS+1% v/v Triton-X100; denoted WB), followed by 2 h blocking at roomtemperature (RT) with 200 μl/well of blocking buffer (PBS+1% w/v BSA).Plates are then washed 3x with WB, followed by 1 h incubation at 37° C.with 50-70 μl/well of added human (or rabbit or sheep) sera (serialdilutions ranging from 1:5-1:250 in dilution buffer (PBS+1% v/vTriton-X100+1% w/v BSA; denoted DB)). Plates are then washed 6x with WB,followed by 1 h incubation at RT with 70 μl/well of AlkalinePhosphatase-conjugated Protein G (3 μg/ml in DB; Calbiochem 539305) orgoat anti-mouse IgG biotin (1 μg/ml, Southern Biotech, 1030-08. In caseof the goat anti-mouse IgG biotin, the plates were washed one extra stepas described, before addition of 100 μlStreptavidin-Alkaline-Phosphatase (1 μg/ml, Sigma Aldrich, S289) andincubated 1 hour at RT. Plates are then washed 6x with WB, followed by10-60 min incubation at room temperature with 100 μl/well of 0.3% w/v ofPhenophtalein monophosphate (Sigma P-5758). Plates are finally quenchedby adding 100 μl/well of Quench solution (0.1M TRIS+0.1M EDTA+0.5MNaOH+0.01% w/v NaN₃; pH14), followed by a measurement with a ELISAreader (ASYS UVM 340) at 550 nm. The strength of the sera, i.e. themagnitude of the humoral immune response, is then reported as thedilution of sera that result in the described Optical Density (OD)value, or the OD value at the indicated dilution of sera.

Example 32 Clinical Trial Protocol

Phase I/IIa Study to Evaluate the Effect of Therapeutic HIV-1Immunization using Vacc-4x+rhuGM-CSF, and HIV-1 Reactivation usingRomidepsin, on the Viral Reservoir in Virologically Suppressed HIV-1Infected Adults on cART.

The primary objective is to measure the effect of treatment withVacc-4x+rhuGM-CSF and cyclic romidepsin treatment on the HIV-1 latentreservoir in HIV-infected patients virologically suppressed on cART.

Endpoints: Primary Endpoints:

1) Safety and tolerability evaluation as measured by adverse events(AE), adverse reactions (AR), serious adverse events (SAE), seriousadverse reactions (SAR), serious unexpected adverse reactions (SUSAR)2) Latent reservoir size measured in CD4+ T cells by:a) HIV-1 viral outgrowth assay (HIV-1 RNA per 106 in resting memory CD4+T cells (RUPM))b) Integrated HIV-1 DNA (copies per 106 CD4+ T cells)c) Total HIV-1 DNA (copies per 106 CD4+ T cells)

Secondary Endpoints PART B

1) Time to re-initiation of cART2) Time to detectable viremia during cessation of cART3) HIV transcription measured as cell associated unspliced HIV-1 RNA(copies per 10⁶ CD4+ T cells)4) HIV-specific T-cell responses as measured by ELISpot, proliferationand/or intracellular cytokine staining5) Plasma HIV-1 viral load6) Histone H3 acetylation as measured in lymphocytes7) T cell count and phenotype8) Antibody titer to Vacc-4x peptides and to p24 as measured by ELISA.

An Open Phase I/IIa Study to Evaluate the Effect of Therapeutic HIV-1Immunization using Vacc-4x+rhuGM-CSF, and HIV-1 Reactivation usingRomidepsin, on the Viral Reservoir in Virologically Suppressed HIV-1Infected Adults on cART. The study is conducted to evaluate thesafety/tolerability of Vacc-4x+rhuGM-CSF as adjunctive therapy toromidepsin and to assess the impact on the latent HIV reservoir and theability to control viral load during an Analytical TreatmentInterruption (n=20, ie. 20 patients).

Target Population: Virologically suppressed (pVL <50 copies/mL) HIV-1infected adults currently on cART.

Study Procedures/Frequency:

1. A pre-treatment phase of 4 weeks (visit 1 to visit 2) to confirm thestability of the latent HIV-1 reservoir and determine baseline HIV-1 Tlymphocyte specific immunity.2. A therapeutic HIV-1 immunization phase of 12 weeks (from visit 2 tovisit 7) in which Vacc-4x will be administered together with rhuGM-CSFat visit 2, 3, 4, 5, 6 and 7 follow by a follow-up period of 2 weeks(visit 8-visit 9).3. A viral reactivation phase of 3 weeks (visit 10-visit 12) consistingof one cycle of romidepsin infusions at a dosing of 5 mg/m2.4. A post-treatment observation phase of ˜8 weeks (visit 13-visit 14) toassess the effect of the investigational treatment on the size of thelatent HIV-1 reservoir.5. An Analytical Treatment Interruption phase of 16 weeks (from aftervisit 15-34).

Investigational Medicinal Products:

Vacc-4x: 1.2 mg administered intradermally at day 0, 7, 14, 21, 77 and84 (visit 2, 3, 4, 5, 6 and 7)rhuGM-CSF: Leukine® (Sanofi) 0.06 mg administered intradermally, 10 minprior to Vacc-4x administration, at day 0, 7, 14, 21, 77 and 84 (visit2, 3, 4, 5, 6 and 7)Romidepsin: Istodax® (Celgene) 5 mg/m2 administered by 3 intravenousinfusion in three consecutive weeks (day 105, 112 and 119) (visit 10,11b and 12) (corresponding to one 28 day cycle).

Trial Design:

1. A pre-treatment phase of 4 weeks (visit 1 to visit 2) to confirm thestability of the latent HIV-1 reservoir and determine baseline HIV-1 Tlymphocyte specific immunity.2. A therapeutic HIV-1 immunization phase of 12 weeks (2 to visit 7) inwhich Vacc-4x will be administered together with rhuGM-CSF at visit 2,3, 4, 5, 6 and 7 followed by a follow-up period of 2 weeks (visit 8 tovisit 9).3. A viral reactivation phase of 3 weeks (visit 10 to visit 12)consisting of one cycle of romidepsin infusions at a dosing of 5 mg/m2.4. A post-treatment observation phase of ˜8 weeks (visit 13 to visit 14)to assess the effect of the romidepsin on the size of the latent HIV-1reservoir.5. An Analytical Treatment Interruption phase of 16 weeks (visit 15-34).

Treatment Vacc-4x

Vacc-4x, consists of four synthetic peptides (Vacc-10 acetate, Vacc-11acetate, Vacc-12 acetate, and Vacc-13 acetate), each corresponding toconserved domains on the HIV-1 p24 capsid protein representing thenative Gag regions with residues 166-185, 252-269, 264-284, and 335-354,respectively.Vacc-4x is manufactured in accordance with Good Manufacturing Practice(GMP) (ref. Vacc-4x IMPD). Vacc-4x is supplied as sterile vials offreeze-dried white powder. There is no additional ingredient in theproduct.RhuGM-CSF (sargramostim, Leukine®, Sanofi)

Leukine® is manufactured by Sanofi and supplied by Genzyme. It is aglycoprotein of 127 amino acids characterized by three primary molecularspecies having molecular masses of 19,500, 16,800 and 15,500 daltons.The liquid Leukine® presentation is formulated as a sterile, preserved(1.1% benzyl alcohol), injectable solution (500 mcg/mL) in a vial.

Lyophilized Leukine® is a sterile, white, preservative-free powder (250mcg) that requires reconstitution with 1 mL Sterile Water for Injection,USP or 1 mL Bacteriostatic Water for Injection, USP. Liquid Leukine® hasa pH range of 6.7-7.7 and lyophilized Leukine® has a pH range of7.1-7.7.

For further information refer to IB (Leukine® prescribing information).

Romidepsin (Istodax®, Celgene)

Istodax® is manufactured by Celgene Corporation. This histonedeacetylase (HDAC) inhibitor is a bicyclic depsipeptide. At roomtemperature, romidepsin is a white powder and is described chemically as(1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-bis(1-methylethyl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone. The empirical formula isC24H36N4O6S2. Istodax® is supplied as a kit containing two vials.Istodax® (romidepsin) for injection is a sterile lyophilized whitepowder and is supplied in a single-use vial containing 10 mg romidepsinand 20 mg povidone, USP. Diluent for Istodax® is a sterile clearsolution and is supplied in a single-use vial containing a 2-mLdeliverable volume. Diluent for Istodax® contains 80% (v/v) propyleneglycol, USP and 20% (v/v) dehydrated alcohol, USP.

For further information refer to IB for romidepsin.

Vacc-4x

Each dose of Vacc-4x (0.1 mL of a 12 mg/mL solution), will beadministered by intradermal injections following the intradermaladministration of rhuGM-CSF (Leukine®) as adjuvant. A total of 6Vacc-4x/rhuGM-CSF immunizations (visit 3, 4, 5, 6, 7 and 8) are plannedin the HIV-1 therapeutic vaccination phase.

Approximately 10 minutes before each administration of Vacc-4x,rhuGM-CSF will be administered intradermally as an adjuvant. Vacc-4xmust be administered intradermally at the same site as rhuGM-CSF,superficial to the deltoid muscle and in the same arm during the courseof the study.

When administering the intradermal injection, utmost care must be takenso that no material is injected subcutaneously. If administeredcorrectly, after puncture of the skin a small bleb should appearfollowing the injection of only a small amount of product. An injectionthat is too superficial should be avoided as this will result in loss ofthe sample volume from the injection site during injection or afterwithdrawal of the needle.

RhuGM-CSF

Each dose of rhuGM-CSF (0.1 mL of 0.60 mg/mL solution) will beadministered as an adjuvant by intradermal injection 10 minutes prior tothe intradermal administration of Vacc-4x immunizations (visit 3, 4, 5,6, 7 and 8) during the HIV-1 therapeutic vaccination phase. rhuGM-CSFmust be administered intradermally at the same site as Vacc-4x,superficial to the deltoid muscle and in the same arm during the entirecourse of the study.

When administering the intradermal injection, utmost care must be takenso that no material is injected subcutaneously. If administeredcorrectly, after puncture of the skin a small bleb should appearfollowing the injection of only a small amount of product. An injectionthat is too superficial should be avoided as this will result in loss ofthe sample volume from the injection site during injection or afterwithdrawal of the needle.

Romidepsin

The dose is 5 mg/m2 administered intravenously over a 4 hour period onDays 1, 8, and 15 of a 28-day cycle (visit 10, 11 and 12).

Trial Assessment: Laboratory Assessment Biochemistry:

Routine biochemistry includes haematology parameters (haemoglobin, totaland differential leukocyte count, platelet count), ALAT, bilirubin,alkaline phosphatase, creatinine, sodium, potassium, phosphorus,magnesium, calcium, urea, albumin and CRP.

HIV Virology:

HIV-1 viral outgrowth (HIV-1 RNA per 10⁶ resting memory CD4+ T cells(RUPM)): The gold standard assay used to measure the frequency ofresting CD4+ T cells carrying latent but replication competent virus isbased on co-culture of highly purified resting CD4+ T cells from thepatient together with PBMCs from an HIV-negative donor and is measuredas infectious units per million cells (IUPM) [Finzi 1999, Chun 2007].

Integrated HIV-1 DNA (copies per 10⁶ CD4+ T cells): Within infectedcells, HIV DNA can exist as linear non-integrated forms, circular formsand as an integrated provirus. In patients receiving effective cART, themajority of HIV DNA is integrated in resting latently infected CD4+ Tcells. The most widely used technique to quantify the number of cellsthat contain integrated virus is the Alu-LTR PCR assay [Sonza 1996].

Total HIV-1 DNA (copies per 10⁶ CD4+ T cells): Total HIV DNA quantifiesintegrated and non-integrated DNA as well as latent and defective virus.There is a strong correlation between total HIV DNA and integrated HIVDNA in patients on cART and therefore cell-associated HIV DNA is likelyto be a good surrogate marker of the total number of latently infectedcells [Koelsch 2008].

Unspliced HIV-1 RNA (copies per 10⁶ CD4+ T cells): HIV transcription ismeasured as copies of cell-associated unspliced HIV-1 RNA/106 CD4+ Tcells using digital droplet PCR Plasma HIV-1 RNA detection by NATscreen: Measured by a transcription mediated amplification (TMA)-basedmethodology, usually referred to as a nucleic acid test (NAT)-screen(PROCLEIX ULTRIO Plus, Genprobe).

Plasma HIV RNA, quantitative viral load: Measured by Roche VL (routineclinical assay) Histone H3 acetylation: Measured in lymphocytes usingflow cytometry with intracellular cytokine stain on fresh isolatedPBMCs.

T Cell count (CD4 and CD8)

Phylogenetic Analysis Immunology:

HIV-Specific T Cell Response as Measured by ELISpot, Proliferationand/or Intracellular Cytokine Staining

Example 33 ELISA Assay to Determine Levels of Antibodies Against HIVEnvelope Glycoprotein gp120 and/or gp41 Buffers Coating Buffer

3.18 g Na₂CO₃ 5.88 g NaHCO₃ Purified water to 1 litre, pH 7.5

Buffer 1

 8.18 g NaCl 0.201 g KCl  2.12 g Na₂HPO₄ × 7H₂O 0.204 g KH₂PO₄ Purifiedwater to 1 litre

Blocking Buffer

  2 L Buffer 1  20 g Bovine serum albumin (BSA)  19 mL 1% NaN₃ 0.2 mLAntifoam pH 7.5

Washing Buffer

7.948 g NaCl 0.201 g KCl  2.1 g Na₂HPO₄ × 7H₂O  0.2 g KH₂PO₄  9.5 mL 1%NaN₃  10.0 mL Triton-X-100  0.1 mL Antifoam Purified water to 1 litre,pH 7.5

Dilution Buffer

20 g BSA 19 mL 1% NaN₃ 0.2 mL Antifoam 20 mL Triton-X-100 Buffer 1 to 2L

Stop-Dilution Solution

24.23 g Trizma Base 58.45 g EDTA 40.00 g NaOH   19 mL 1% NaN₃ Purifiedwater to 2 L

Vacc-C5:

Dimeric peptide, which consists of C5 domain of HIV gp1 20 with thetransmembrane domain of gp41 and/or with the constant C2 domain of gp120with the following structure:

(H-Glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-glycyl-glycyl-L-cysteinyl(2-oxo-ethyl)-glycyl-glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-L-glutaminyl-L-arginyl-L-glutamyl-L-lysyl-L-arginyl-L-alanyl-glycyl-L-glutamyl-L-arginyl-L-glutamyl-L-lysyl-L-arginyl-L-alanyl-NH₂)(H-Glycyl-L-lysyl-glycyl-glycyl-L-isoleucyl-L-glutamyl-L-glutamyl-L-glutamyl-glycyl-glycyl-L-arginyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-glycyl-glycyl-L-glutaminyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-NH₂), acetate salt (amide bond between Cys(2-oxo-ethyl)¹⁰ (A-chain) andLys² (B-chain))

This compound may also be referred to as:

(H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys(2-oxo-ethyl)-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH₂)(H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH₂),acetate salt (amide bond between Cys(2-oxo-ethyl)¹⁰ (A-chain) and Lys²(B-chain))

Vacc-C5 consists of two linear peptide amide chains with 31 amino acids(A-chain) and 22 amino acids (B-chain). Each chain has a free aminogroup at the N-terminus and an amide group at the C-terminus. The chainsare covalently linked via an amide bond between Cys(2-oxo-ethyl)¹⁰ ofthe A-chain and Lys² of the B-chain. All amino acid residues except theachiral Gly are in the L-configuration.

Method

ELISA plates (Nunc 249946, conical bottomed) were coated with Vacc-C5 ata final concentration of 16 μg/mL in coating buffer for 4 days at 4° C.,washed three times with washing buffer (WB) and stored dried at 4° C.until required. Blocking buffer (200 μL per well) was added and theplates were incubated at room temperature for 2 hours before washingthree times with WB. Serum samples were diluted using dilution bufferand stored at 2-8° C. for up to a maximum of 24 hours. Samples (100 μL)were added to the appropriate wells of the ELISA plate(s) and incubatedat 37° C. for 2 hours. After washing the plates six times with WB, thebound antibody was detected using a Protein G-alkaline phosphataseconjugate (Calbiochem 539305), which binds immunoglobulin, and thesubstrate phenolphtalein monophosphate. The conjugate was diluted and100 μL added to each well at a concentration of 500 μg/mL. The plateswere incubated for 1 hour at room temperature before washing six timeswith WB. The substrate was added (100 μL per well) and incubated for 15minutes at room temperature before colour development was stopped by theaddition of stop-dilution solution. Colour changes of the substrateoccur in proportion to the amount of bound serum antibody and values arereported as optical density (OD) readings taken at 550 nm/620 nm.

Each assay included a sheep anti-HIV-1 positive control serum (D7324;Aalto, Dublin, Ireland) raised against the C5 region of gp120. Theconcentration of anti-05 antibody in the test samples was determined bycomparison to a standard curve prepared using dilutions of the positivecontrol. As the positive control anti-05 antibodies will not beidentical to the anti-C5 antibodies present in HIV-infected individualsthe ELISA is regarded a semi-quantitative immunoassay. Serum samples arepreferred for this assay, but plasma samples could be used withcomparable results.

The samples were initially screened in duplicate at a 5x dilution and ifthe OD value was outside the linear part of the standard curve (i.e.,OD>0.8) the plasma samples were diluted further and assayed again untilan OD value within the linear range was obtained. If more than one valuewas within the linear range, the lowest dilution of sample (normallywith an OD value of around 0.5) was used to calculate the concentrationof anti-05 antibodies in the sample under test. Sera from uninfectedblood donors were used to determine a cut off value for negativeresponse of OD_(550/620)=0.1 (equal to three times the observed mean OD550/620).

In order to determine the region(s) of the Vacc-C5 sequence recognisedby antibodies in the plasma samples, blocking experiments wereconducted. Plasma samples were diluted in dilution buffer and incubatedovernight at 2-8° C. with either 100 or 500 μM of peptide. The dilutionfactor used for each plasma sample was calculated to give a value in thelinear range for the assay. The blocking peptides used were eithercomplete Vacc-C5 or shorter peptide sequences from within the C5 regionof gp120 (peptides 105, 104 and 015) or within the adjacent gp41 region(peptide 201). The amino acid sequences for the shorter peptidesequences were derived from the HIV-1 wild-type consensus sequence(www.hiv.lanl.gov) The sequences of the peptides used in the blockingassays are shown in Error! Reference source not found. below and thelocation of the peptides within the Vacc-C5 antigen is showndiagrammatically in Error! Reference source not found.

OD values in the absence of peptide (phosphate-buffered saline (PBS)control) were taken as 100% and the percentage of blocking by eachpeptide calculated against this value.

Subtraction from 100 gave the % inhibition for each peptide in thesample. Negative values were set to zero.

TABLE 1 Amino acid sequences of Vacc-C5 and overlapping peptides.Peptide Sequence ¹Vacc-C5 gp120: GAKRRVVGG C GGAKRRVVQREKRAGEREKRA         | gp41:         G K GGIEEEGGRDRDRGGQDRDR BI400-105 gp120:   AKRRVV BI400-104 gp120:     RVVQREK BI400-015 gp120:  APTKAKRRVVQREKR BI400-201 gp41:  DRPEGIEEEGGERDR ¹The two peptidescontained within Vacc-C5 are linked by a thioether bond between theside-chains of the marked cysteine and lysine residues.

Results:

Patients in the Phase II Study CT-BI Vacc-4x 2007/1 (described inexample 25) with high levels of pre-existing antibodies against adefined region of HIV envelope glycoprotein gp120 and/or gp41 asmeasured be the ELISA assay described above in this example (33) andsubsequently treated with Vacc-4X had a significant lower median viralload (VL) set-point as compared to patients treated with placebo. Thedata indicates that the higher amount of antibodies against a specificregion of HIV envelope glycoprotein gp120 and/or gp41, the lower was themedian viral load set-point and the larger the difference to the grouptreated with placebo.

Serum samples from patients in the Phase II Study CT-BI Vacc-4x 2007/1(N=115) at different time points (week 1, 28 and 52) were analysed forantibodies against Vacc-C5.

The effect of the level of pre-existing antibodies against Vacc-C5 atWeek 1 (prior to Vacc-4x treatment and ART interruption) on median ViralLoad (VL) was analyzed for ‘Subgroup D’ of the clinical study (LOCFpopulation, patients that resumed ART after Week 40 that in addition hada pre-ART viral load value available). The results are summarized inTable 2.(N=71).

TABLE 2 C5 Ab VL set-point Pre-ART VL conc. Treatment Median Mean MedianMean Viral load values in subgroup D and antibody cut-off at 2 μg/ml <2Vacc-4x 40050 71010 65584 227689 μg/ml Placebo 74425 86452 40365112617 >2 Vacc-4x 18183 31711 48290 125047 μg/ml Placebo 69850 7442842636 49827 Viral load values in subgroup D and antibody cut-off at 5μg/ml <5 Vacc-4x 39650 65991 60470 209164 μg/ml Placebo 68650 8289052940 110993 >5 Vacc-4x 9115 27707 78968 135710 μg/ml Placebo 10755084134 32750 42792 Viral load values in subgroup D and antibody cut-offat 10 μg/ml <10  Vacc-4x 39650 63683 62350 214204 μg/ml Placebo 6865079955 52940 105387 >10  Vacc-4x 5520 18723 49180 50623 μg/ml Placebo111500 103550 32750 32831

Example 34 Alternative ELISA Assay to Determine Levels of AntibodiesAgainst HIV Envelope Glycoprotein Gp120 and/or Gp41, and its Applicationto a Comparison of Matrix Effects Preparation of Buffers and WorkingSolutions 5 M Hydrochloric Acid

Add approximately 500 mL of ultra-high purified water to a 1 litrevolumetric flask. Slowly add 182.3 mL of hydrochloric acid. Make up to 1litre with ultra-high purified water and mix gently by inversion. Storefor up to 1 year at room temperature.

5 M Sodium Hydroxide

This is an exothermic reaction. Add 750 mL of ultra-high purified waterto a 2 litre volumetric flask. Store this flask in a room temperaturewater bath. Slowly add 200 g of NaOH to the flask. During this processstir the solution with a glass rod. Allow the solution to cool down andthen make up to 2 litres with ultra-high purified water and mix gentlyby inversion. Store for up to 1 year at room temperature.

Sodium Azide (10% w/v)

Sodium azide must be handled in a fume cupboard. Dissolve 1 g of sodiumazide in water to give a final concentration of 10% sodium azide. Mixgently by inversion ensuring the sodium azide is fully dissolved. Storeat 2-8° C. for up to 1 year.

Working Conjugate Solution 3.00 μg/mL

72 μL of conjugate+11,928 μL of dilution buffer. Mix gently for at least30 minutes prior to use (using a rotamixer).

Coating Buffer pH 9.5±0.1

Add 900 mL of water to a 1 litre volumetric flask. Then add:3.18 g ofNaCO₃, 5.88 g of NaHCO3. Mix using a magnetic stirrer and adjust pH to9.5±0.1 using 5 M Sodium hydroxide/5 M Hydrochloric acid solution. Makeup to 1 litre with ultra-high purified water.

Blocking Buffer

20 g of BSA, 1 PBS tablet, 200 mL of water, Mix using a magneticstirrer.

Dilution Buffer

1 PBS tablet, 2 g BSA, 2 mL Triton X100, 200 mL water. Mix using amagnetic stirrer.

Washing Buffer

5 PBS tablet, 1000 mL water, 1 mL Triton X 100. Mix using a magneticstirrer.

Stop Solution

Add the following to a 2 litre volumetric flask: 24.23 g Trizma Base,58.45 g EDTA, 40.00 g NaOH, 19 mL of 1% NaN₃, Add 1.8 liters of water.Mix using a magnetic stirrer. Make up to 2 litres using ultra-highpurified water. Measure pH (acceptable value range 12-13). Expires after1 year.

Primary Positive Control Standard Stock (2.0 mg/mL)

Reconstitute Sheep anti-HIV-1 gp120 antibody raised against the C5region of gp120 (D7324; Aalto, Dublin, Ireland) with 1 mL of ultra-highpurified water. Mix gently using a vortex mixer ensuring material hasbeen resuspended. Centrifuge for 5 minutes at 3000 rpm. Separate theresulting supernatant into 60 μL aliquots and store at −20° C. forfuture use.

Secondary Positive Control Standard Stock (100 μg/mL)

50 μL of primary positive control+950 μL dilution buffer. Mix by vortexmixer. Use on day of preparation.

Working Standards

Dilute secondary stock 1:20 with dilution buffer, and then by 2:1 serialdilution (5000-78 ng/ml range).

Method

ELISA plates (Nunc 249946, conical bottomed) were coated with Vacc-C5 ata final concentration of 16 μg/mL in coating buffer for 4 days at 4° C.,washed three times with washing buffer (WB) and stored dried at 4° C.until required.

Immunoassay Procedure

1. Remove from storage the number of dried plates or strips that areneeded for the assay.2. Add washing buffer 300 μL per well then aspirate. Repeat twice more(3 times in total). Aspirate the last wash and blot the plate dry byinverting over absorbent towel.3. Add 200 μL blocking buffer to each well. Cover with plate sealer andincubate at room temperature for 2 hours, no shaking.4. Dilute all matrix samples minimally 1 in 5 with dilution buffer (asper section 10).5. Wash 3 times (as per step 2), with washing buffer, 300 μL per well.Remove all liquid from the wells by inverting over absorbent towel.6. Add 100 μL of controls, standards and samples to coated plate induplicate.7. Cover with plate sealer and incubate at 37° C. for 2 hours in a dampbox.8. Prepare protein G/AP (working conjugate) dilution at a concentrationof 3.00 μg/mL. Perform preparation at least 30 minutes prior to use.9. Wash 6 times with washing buffer, 300 μL per well. Remove all liquidfrom the wells by inverting over absorbent towel.10. Add 100 μL protein G/AP solution to each well, cover with platesealer and incubate at room temperature for 1 hour, no shaking.11. Wash 6 times (as per step 2) with washing buffer, 300 μL per well.Remove all liquid from the wells by inverting over absorbent towel.12. Add 100 μL of pNPP solution to each well. Cover with plate sealerand incubate at room temperature for 15 minutes, no shaking.13. Add 200 μL stop solution to each well. If the colour is green ordoes not appear uniform within the well, gently shake the plate to mixthoroughly.14. Measure the absorbance at 405 nm, reference 540 nm.15. Process raw data, using a 5PL (auto-estimate) algorithm, with aweighting factor of 1/Y.

Vacc-C5:

Dimeric peptide, which consists of C5 domain of HIV gp120 with thetransmembrane domain of gp41 and/or with the constant C2 domain of gp120with the following structure:

(H-Glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-glycyl-glycyl-L-cysteinyl(2-oxo-ethyl)-glycyl-glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-L-glutaminyl-L-arginyl-L-glutamyl-L-lysyl-L-arginyl-L-alanyl-glycyl-L-glutamyl-L-arginyl-L-glutamyl-L-lysyl-L-arginyl-L-alanyl-NH₂)(H-Glycyl-L-lysyl-glycyl-glycyl-L-isoleucyl-L-glutamyl-L-glutamyl-L-glutamyl-glycyl-glycyl-L-arginyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-glycyl-glycyl-L-glutaminyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-NH₂), acetate salt (amide bond between Cys(2-oxo-ethyl)¹⁰ (A-chain) andLys² (B-chain))

This compound may also be referred to as:

(H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys(2-oxo-ethyl)-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH₂)(H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH₂),acetate salt (amide bond between Cys(2-oxo-ethyl)¹⁰ (A-chain) and Lys²(B-chain))

Vacc-C5 consists of two linear peptide amide chains with 31 amino acids(A-chain) and 22 amino acids (B-chain). Each chain has a free aminogroup at the N-terminus and an amide group at the C-terminus. The chainsare covalently linked via an amide bond between Cys(2-oxo-ethyl)¹⁰ ofthe A-chain and Lys² of the B-chain. All amino acid residues except theachiral Gly are in the L-configuration.

Results

To investigate the effect of different matrices the concentrations ineight human serum samples spiked at low and ten human plasma samplesspiked at low (2500 ng/mL) and high (10000 ng/mL) concentrations ofeither Sheep anti-HIV-1 gp120 antibody raised against the C5 region ofgp120 (D7324; Aalto, Dublin, Ireland), or biobank samples withpreviously determined concentration. Matched unspiked samples were alsoanalyzed to confirm absence of background. The recovery was found to becomparable between spiked serum and plasma samples (summary tablebelow).

Matrix Serum (N = 8) Plasma (N = 10) Spike Sheep Ab Biobank Sample SheepAb Biobank Sample Target conc. (ng/ml) 2500 10000 2500 10000 2500 100002500 10000 Mean recovery (%) 81 71.8 80.9 73.6 82 80.4 83 91.8 SD (%)18.1 10.7 21.7 12.4 16.8 10.9 4.7 12.8

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofproduct, composition, process, or use claims and may include, by way ofexample and without limitation, the claims.

1. A method for reducing and/or delaying one or more pathologicaleffects of human immunodeficiency virus I (HIV) or for reducing the riskof developing acquired immunodeficiency syndrome (AIDS) in a humaninfected with HIV, the method comprising the steps of: a) measuring in abiological sample, such as serum or plasma, from a human infected withHIV the amount of antibodies against one or more epitope of HIV envelopeglycoproteins gp120 and/or gp41 in a suitable assay; b) selecting asubgroup of humans from a), wherein the amount of said measuredantibodies corresponds to an amount of above background level ofuninfected humans, such as above 1 μg/ml of antibodies against Vacc-C5in serum as measured by an ELISA assay as described in example 33; c)treating said humans infected with HIV selected under b) with one ormore peptide(s) to stimulate a cell-mediated immune response and/or acompound that stimulates a humoral response in said human.
 2. (canceled)3. The method according to claim 1, wherein said one or more peptidethat stimulate a Cell-mediated immune response is at least oneHIV-specific peptide comprising an amino acid sequence selected from thegroup of: (SEQ ID NO: 47) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Ala Xaa₈ Xaa₉Gln Thr Pro Trp Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Val Xaa₂₀;

wherein Xaa in position 1 is Lys or Arg, Xaa in position 2 is Ala, Gly,Ser or Arg, Xaa in position 3 is Leu or Met, Xaa in position 4 is Gly orArg, Xaa in position 5 is Pro, Thr, Val, Ser, Gln or Ala, Xaa inposition 6 is Gly, Ala, Lys, Arg, Gln or Glu, Xaa in position 8 is Thror Ser, Xaa in position 9 is Leu or Be, Xaa in position 14 is Thr, Seror Val, Xaa in position 15 is Ala or Ser, Xaa in position 16 is Cys orSer, Xaa in position 17 is Gln or Leu, Xaa in position 18 is Gly, Glu orArg, and Xaa in position 20 is Gly or Arg; (SEQ ID NO: 50) Xaa₁ Xaa₂Xaa₃ Xaa₄ Xaa₅ Gly Leu Asn Pro Leu Val [Gly]_(n) Xaa₁₂ Xaa₁₃ Tyr Xaa₁₅Pro Xaa₁₇ Xaa₁₈ Ile Leu Xaa₂₁ Xaa₂₂;

wherein Xaa in position 1 is Arg, Lys, Asp or none, Xaa in position 2 isTrp, Gly, Lys or Arg, Xaa in position 3 is Be, Leu, Val or Met, Xaa inposition 4 is Be, Val or Leu, Xaa in position 5 Leu, Met, Val or Pro,Xaa in position 12 is Arg or Lys, Xaa in position 13 is Met or Leu, Xaain position 15 is Ser, Cys or Gln, Xaa in position 17 is Thr, Val, Be,Ser or Ala, Xaa in position 18 is Ser, Gly or Thr, Xaa in position 21 isAsp, Glu, Cys or Gly, Xaa in position 22 is Gly or none, and n=0, 1, 2or 3; (SEQ ID NO: 55) Xaa₁ Xaa₂ Xaa₃ Pro Ile Pro Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ [Gly]_(n) Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄;

wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or none,Xaa in position 2 is Asn, Ala or Lys, Xaa in position 3 is Pro, Gln,Gly, Ile or Leu, Xaa in position 7 is Val or Ala, Xaa in position 8 isGly or Lys, Xaa in position 9 is Glu, Asp, Lys, Phe or Thr, Xaa inposition 10 is Be, Met, Val or Leu, Xaa in position 11 is Tyr, Leu ornone, Xaa in position 12 is Ser or none, Xaa in position 13 is Arg ornone, Xaa in position 14 is Asp, Arg, Trp, Ala or none, Xaa in position15 is Be or none, Xaa in position 16 is Tyr or none, Xaa in position 17is Lys or Arg, Xaa in position 18 is Arg, Lys or Asp, Xaa in position 19is Trp or Gly, Xaa in position 20 is Be, Met, Val, Gln or Ala, Xaa inposition 21 is Be, Val or Ala, Xaa in position 22 is Leu, Met or Val,Xaa in position 23 is Gly or Cys, Xaa in position 24 is Leu or none,n=1, 2 or 3; and (SEQ ID NO: 61) Xaa₁ Xaa₂ Ile Ile Xaa₅ Xaa₆ Xaa₇ Xaa₈Xaa₉ Leu Xaa₁₁ [Gly]_(n) [Arg]_(m) Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅;

wherein Xaa in position 1 is Pro, Lys, Arg or none, Xaa in position 2 isGlu, Arg, Phe or Lys, Xaa in position 5 is Pro or Thr, Xaa in position 6is Met, Thr or Nleu, Xaa in position 7 is Phe or Leu, Xaa in position 8is Ser, Thr, Ala or Met, Xaa in position 9 is Ala, Glu or Leu, Xaa inposition 11 is Ser or none, Xaa in position 12 is Ala, Arg or none, Xaain position 13 is Be, Leu or none, Xaa in position 14 is Ser, Ala, Leuor none, Xaa in position 15 is Tyr, Glu or Asp, Xaa in position 16 isGly or Asp, Xaa in position 17 is Ala or Leu, Xaa in position 18 is Thr,Be, Val, Leu or Asn, Xaa in position 19 is Pro, Thr or Ser, Xaa inposition 20 is Tyr, Phe, Nleu, His or Gln, Xaa in position 21 is Asp,Asn, Leu or Ala, Xaa in position 22 is Leu, Ile, Val or Asn, Xaa inposition 23 is Asn, Tyr, Cys or Gly, Xaa in position 24 is Thr, Met,Ile, Ala, Val or none, Xaa in position 25 is Gly or none, n=1, 2 or 3and m=0, 1, 2 or 3 independent of each other; wherein the terminal endsof each HIV specific peptide may be free carboxyl- or amino-groups,amides, acyls or acetyls or salts thereof, such as wherein each peptideis in the form of an acetate salt. 4-17. (canceled)
 18. The methodaccording to claim 3, wherein said compound that stimulates a humoralresponse in a subject is an agent capable of stabilising the associationof the C5 domain of HIV gp120 with the transmembrane domain of gp41and/or with the constant C2 domain of gp120 or which compound inducesantibodies that stabilise association of the C5 domain of HIV gp120 withthe transmembrane domain of gp41 and/or with the constant C2 domain ofgp120.
 19. The method according to claim 3, wherein the at least onecompound that stimulates a humoral response stabilising association ofthe C5 domain of HIV gp120 with the transmembrane domain of gp41 and/orwith the constant C2 domain of gp120 is a molecule comprising at leastone amino acid sequence selected independently from an amino acidsequence derived from the transmembrane domain of gp41 and an amino acidsequence derived from the C2 domain, wherein the at least one amino acidsequence binds the C5 domain, optionally comprising at least one D-aminoacid. 20-41. (canceled)
 42. The method according to claim 1, whichmethod further comprises the administration of an immunomodulatorycompound and/or a reservoir purging agent, and/or an adjuvant. 43.(canceled)
 44. The method according to claim 42, wherein the adjuvant,is recombinant human granulocyte-macrophage colony-stimulating factor(rhuGM-CSF).
 45. The method according to claim 42, which method furthercomprises the administering of an immunomodulatory compound such as animmunomodulatory compound selected from anti-PD1 antibodies, such asMDX-1106 (Merck), THALOMID® (thalidomide), anti-PD1 antibodies,cyclophosphamide, Levamisole, lenalidomide, CC-4047 (pomalidomide),CC-11006 (Celgene), and CC-10015 (Celgene), and immunomodulatorycompound described in any one of WO2007028047, WO2002059 106, andWO2002094180, and a4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione, and a3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. 46-48.(canceled)
 49. The method according to claim 42, wherein the reservoirpurging agent is, a histone deacetylase (HDAC) inhibitor. 50-55.(canceled)
 56. A method for reducing and/or delaying pathologicaleffects of human immunodeficiency virus I (HIV) or for reducing the riskof developing acquired immunodeficiency syndrome (AIDS) in a humaninfected with HIV, the method comprising the steps of: a) measuring in abiological sample, such as serum or plasma, from a human infected withHIV the amount of antibodies against one or more epitope of HIV envelopeglycoproteins gp120 and/or gp41 in a suitable assay; b) selecting asubgroup of humans from a), wherein the amount of said measuredantibodies corresponds to an amount above background level in uninfectedhumans and below 1 μg/ml of antibodies against Vacc-C5 in serum asmeasured by an ELISA assay as described in Example 33; c) treating saidhumans infected with HIV selected under b) with a compound thatstimulate a humoral response in a subject; d) optionally having amaturation period, such as a period of up to 4 weeks; e) optionallyrepeating the measurement in a) and selecting humans infected with HIV,wherein the amount of said measured antibodies corresponds to an amountof above background level of uninfected humans, such as above 1 μg/ml ofantibodies against Vacc-C5 in serum as measured by an ELISA assay asdescribed in example 33; f) treating said humans infected with HIVselected under b) or e) with one or more peptides to stimulate acell-mediated immune response and/or a compound that stimulate a humoralresponse in said human.
 57. The method according to claim 56, whereinsaid compound that stimulates a humoral response in a subject under c)is an agent capable of stabilising the association of the C5 domain ofHIV gp120 with the transmembrane domain of gp41 and/or with the constantC2 domain of gp120 or which compound induces antibodies that stabiliseassociation of the C5 domain of HIV gp120 with the transmembrane domainof gp41 and/or with the constant C2 domain of gp120.
 58. The methodaccording to claim 57, wherein the at least one agent capable ofstabilising association of the C5 domain of HIV gp120 with thetransmembrane domain of gp41 and/or with the constant C2 domain of gp120is a molecule comprising at least one amino acid sequence selectedindependently from an amino acid sequence derived from the transmembranedomain of gp41 and an amino acid sequence derived from the C2 domain,wherein the at least one amino acid sequence binds the C5 domain,optionally comprising at least one D-amino acid. 59-81. (canceled) 82.The method according to claim 58, wherein said one or more peptides thatstimulate a Cell-mediated immune response is at least one HIV-specificpeptide selected from the group of amino acid sequences: (SEQ ID NO: 47)Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Ala Xaa₈ Xaa₉ Gln Thr Pro Trp Xaa₁₄ Xaa₁₅Xaa₁₆ Xaa₁₇ Xaa₁₈ Val Xaa₂₀;

wherein Xaa in position 1 is Lys or Arg, Xaa in position 2 is Ala, Gly,Ser or Arg, Xaa in position 3 is Leu or Met, Xaa in position 4 is Gly orArg, Xaa in position 5 is Pro, Thr, Val, Ser, Gln or Ala, Xaa inposition 6 is Gly, Ala, Lys, Arg, Gln or Glu, Xaa in position 8 is Thror Ser, Xaa in position 9 is Leu or Be, Xaa in position 14 is Thr, Seror Val, Xaa in position 15 is Ala or Ser, Xaa in position 16 is Cys orSer, Xaa in position 17 is Gln or Leu, Xaa in position 18 is Gly, Glu orArg, and Xaa in position 20 is Gly or Arg; (SEQ ID NO: 50) Xaa₁ Xaa₂Xaa₃ Xaa₄ Xaa₅ Gly Leu Asn Pro Leu Val [Gly]_(n) Xaa₁₂ Xaa₁₃ Tyr Xaa₁₅Pro Xaa₁₇ Xaa₁₈ Ile Leu Xaa₂₁ Xaa₂₂;

wherein Xaa in position 1 is Arg, Lys, Asp or none, Xaa in position 2 isTrp, Gly, Lys or Arg, Xaa in position 3 is Be, Leu, Val or Met, Xaa inposition 4 is Be, Val or Leu, Xaa in position 5 Leu, Met, Val or Pro,Xaa in position 12 is Arg or Lys, Xaa in position 13 is Met or Leu, Xaain position 15 is Ser, Cys or Gln, Xaa in position 17 is Thr, Val, Be,Ser or Ala, Xaa in position 18 is Ser, Gly or Thr, Xaa in position 21 isAsp, Glu, Cys or Gly, Xaa in position 22 is Gly or none, and n=0, 1, 2or 3; (SEQ ID NO: 55) Xaa₁ Xaa₂ Xaa₃ Pro Ile Pro Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ [Gly]_(n) Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄;

wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or none,Xaa in position 2 is Asn, Ala or Lys, Xaa in position 3 is Pro, Gln,Gly, Ile or Leu, Xaa in position 7 is Val or Ala, Xaa in position 8 isGly or Lys, Xaa in position 9 is Glu, Asp, Lys, Phe or Thr, Xaa inposition 10 is Be, Met, Val or Leu, Xaa in position 11 is Tyr, Leu ornone, Xaa in position 12 is Ser or none, Xaa in position 13 is Arg ornone, Xaa in position 14 is Asp, Arg, Trp, Ala or none, Xaa in position15 is Be or none, Xaa in position 16 is Tyr or none, Xaa in position 17is Lys or Arg, Xaa in position 18 is Arg, Lys or Asp, Xaa in position 19is Trp or Gly, Xaa in position 20 is Be, Met, Val, Gln or Ala, Xaa inposition 21 is Be, Val or Ala, Xaa in position 22 is Leu, Met or Val,Xaa in position 23 is Gly or Cys, Xaa in position 24 is Leu or none,n=1, 2 or 3; and (SEQ ID NO: 61) Xaa₁ Xaa₂ Ile Ile Xaa₅ Xaa₆ Xaa₇ Xaa₈Xaa₉ Leu Xaa₁₁ [Gly]_(n) [Arg]_(m) Xaa₁₂ Xaa₁₃ Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅;

wherein Xaa in position 1 is Pro, Lys, Arg or none, Xaa in position 2 isGlu, Arg, Phe or Lys, Xaa in position 5 is Pro or Thr, Xaa in position 6is Met, Thr or Nleu, Xaa in position 7 is Phe or Leu, Xaa in position 8is Ser, Thr, Ala or Met, Xaa in position 9 is Ala, Glu or Leu, Xaa inposition 11 is Ser or none, Xaa in position 12 is Ala, Arg or none, Xaain position 13 is Be, Leu or none, Xaa in position 14 is Ser, Ala, Leuor none, Xaa in position 15 is Tyr, Glu or Asp, Xaa in position 16 isGly or Asp, Xaa in position 17 is Ala or Leu, Xaa in position 18 is Thr,Be, Val, Leu or Asn, Xaa in position 19 is Pro, Thr or Ser, Xaa inposition 20 is Tyr, Phe, Nleu, His or Gln, Xaa in position 21 is Asp,Asn, Leu or Ala, Xaa in position 22 is Leu, Ile, Val or Asn, Xaa inposition 23 is Asn, Tyr, Cys or Gly, Xaa in position 24 is Thr, Met,Ile, Ala, Val or none, Xaa in position 25 is Gly or none, n=1, 2 or 3and m=0, 1, 2 or 3 independent of each other; wherein the terminal endsof each HIV specific peptide may be free carboxyl- or amino-groups,amides, acyls or acetyls or salts thereof, such as wherein each peptideis in the form of an acetate salt. 83-93. (canceled)
 94. The methodaccording to claim 82, wherein said one, two, three or four peptideacetate salts are in a dissolved liquid state, such as water. 95.(canceled)
 96. The method according to claim 56, which method furthercomprises the administration of an immunomodulatory compound and/or areservoir purging agent, such as a histone deacetylase (HDAC) inhibitor.97. (canceled)
 98. The method according to any one of claim 56, whichmethod further comprises the administering of an effective amount of anadjuvant, such as recombinant human granulocyte-macrophagecolony-stimulating factor (rhuGM-CSF).
 99. The method according to claim96, which method further comprises the administering of animmunomodulatory compound.
 100. The method according to claim 99,wherein said immunomodulatory compound is selected from anti-PD1antibodies, such as MDX-1106 (Merck), THALOMID® (thalidomide), anti-PD1antibodies, cyclophosphamide, Levamisole, lenalidomide, CC-4047(pomalidomide), CC-11006 (Celgene), and CC-10015 (Celgene), andimmunomodulatory compound described in any one of WO2007028047,WO2002059106, and WO2002094180.
 101. The method according to claim 99,wherein said immunomodulatory compound is selected from a4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione and a3-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-2,6-dione. 102.(canceled)
 103. The method according to claim 96, which method furthercomprises the administering of a reservoir purging agent, such as aHistone deacetylase (HDAC) inhibitor.
 104. The method according to claim42, wherein said reservoir purging agent, such as a Histone deacetylase(HDAC) inhibitor is selected from M344(4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide), chidamide(CS055/HBI-800), 4SC-202, (4SC), Resminostat (4SC), hydroxamic acidssuch as vorinostat (SAHA), belinostat (PXD101), LAQ824, trichostatin Aand panobinostat (LBH589); benzamides such as entinostat (MS-275),CI994, and mocetinostat (MGCD0103), cyclic tetrapeptides (such astrapoxin, such as trapoxin B), and the depsipeptides, such as romidepsin(ISTODAX), electrophilic ketones, and the aliphatic acid compounds suchas phenylbutyrate, valproic acid, Oxamflatin, ITF2357 (genericgivinostat), Apicidin, MC1293, CG05, and CG06; compounds that activatetranscription factors including NF-KappaB, Prostratin, auranofin,bryostatin, a nontumorigenic phorbol ester, DPP(12-deoxyphorbol-13-phenylacetate), PMA, and Phorbol 12-myristate13-acetate (PMA); Compounds that activate HIV mRNA elongation includingP-TEF-b kinase and hexamethylbisacetamide (HMBA); IL-7; T-cellstimulating factors including anti-CD3/CD28− T-cell stimulating Ab's;Kinase inhibitors including Tyrphostin A, Tyrphostin B, and TyrphostinC; PTEN (phosphatase and tensin homologue) gene inhibitors includingSF1670 (Echelon Bioscience), Disulfiram (DSF), an inhibitor ofacetaldehyde dehydrogenase, Protein Tyrosine Phosphatase Inhibitorsincluding bpV(HOpic), bpV(phen), and bpV(pic) (Calbiochem; EMDMillipore), Toll-like receptors agonists including Toll-like receptor-9(TLR9) and Toll-like receptor-7 (TLR9) agonists, quercetin, lipoic acid,sodium butyrate, TNF-alpha, PHA, Tat. 105-111. (canceled)